Ocular formulations for drug-delivery and protection of the anterior segment of the eye

ABSTRACT

The present application relates to topical formulations comprising Compound-I 
                         
or its free base, and a second active agent selected from nicotinic acid, nicotinamide, and vitamin K, and a combination thereof, for treating ocular neovascularization. The present application also relates to pharmaceutical compositions comprising particles of Compound-I or its free base, and suspension formulations comprising the particle compositions of Compound-I or its free base.

RELATED APPLICATION

This application claims priority to, and the benefit of, U.S. Ser. No.62/051,794, filed on Sep. 17, 2014, the contents of which areincorporated by reference in their entirety.

FIELD OF THE APPLICATION

Embodiments disclosed herein are generally directed to topicaladministration of a combination of a first active agent, e.g., apharmaceutical compound or a salt thereof, and a second active agent, totreat ocular diseases or conditions. The embodiments disclosed includeocular formulations comprising a first active agent or a salt thereofand a second active agent, where the formulation is a solution or asuspension. The solution or suspension may further comprise asolubilizing agent, and is suitable for delivering the first activeagent or a salt thereof, to the posterior segment of the eye whileprotecting the anterior segment of the eye of a subject.

BACKGROUND

Treatment of diseases or disorders of the posterior segment of the eyewith topically applied active agents has not been effective because ofinefficient delivery of the active agent to the target site. The vastmajority of topical drugs penetrate via the cornea. However, the corneais not equally permeable to all topically applied active agents, sincethe basic structure of the cornea dictates the relative penetration ofactive agent. Effectively, the greatest barrier to active agentpenetration is the corneal epithelium which is rich in cellularmembranes and is therefore more susceptible to penetration by lipophilicagents. In contrast, since the corneal stroma is largely constituted ofwater, active agents pass through more readily if they are hydrophilic.The endothelium represents a monolayer that, once more, is lipophilic.Active agents which are lipophilic or amphiphilic, in that they canbehave as either charged or non-charged, penetrate the cornea best.Similar to the cornea, the conjunctival epithelium and blood vesselswithin or under the conjunctival epithelium may be penetrated by thesame type of lipophilic or biphasic agents. However, because of thenature of the lipophilic membranes in the conjunctive and its inherentvasculature, most active agents typically do not penetrate through theconjunctiva and into the eye. Agents with limited penetration into thevascular tissues in the conjunctival and subconjunctival regions aredrained into the systemic circulation.

Because of the limited permeability of many topical drops to the cornealand conjunctival barriers, one major disadvantage of topical drops maybe the need for high concentration of active agents in the topicalformulation in order to achieve meaningful therapeutic drug levels ininternal ocular tissues. Depending on the active agent, the moleculeitself or high concentrations thereof, a topical formulation may betoxic to the anterior segment of the eye, including the conjunctiva,cornea and/or lens, causing various injuries to the ocular surface, suchas corneal epithelial defects and erosions.

Ocular side-effects observed following treatment with anti-EGFR drugtherapies, e.g., anti-EGFR cancer therapies, have illuminated theessential role the EGFR signaling pathway plays in maintaining andrestoring the health of the human corneal epithelium. Patients treatedwith anti-EGFR drug therapies can develop corneal changes, such asepithelial degeneration and defects, ulceration, corneal epithelialthinning, erosions and/or corneal edema, keratitis as well asperforation while undergoing therapy or even after discontinuation ofthe anti-EGFR therapy. The important role of EGFR signaling inhomeostasis and pathophysiology of the corneal epithelium is wellestablished. EGFR activation is both necessary and sufficient forcorneal epithelial migration, proliferation, and differentiation.Moreover, EGFR is the primary mediator of wound healing during in vitroexperiments with immortalized human corneal epithelial cells. Therefore,treating ocular diseases or disorders (e.g., diseases or disorders ofthe posterior segment of the eye) would benefit from administration ofan agent that maintains EGFR activity in addition to the administrationof a formulation comprising an active agent (e.g., an active agenthaving toxic or anti-EGFR activity).

The present application provides novel formulations which circumvent theproblems encountered in ocular delivery of existing topical therapeuticagents. The present application accomplishes the combined effects ofdecreasing corneal and anterior segment drug exposure and protectingcorneal and anterior segment tissues, while increasing posterior segmentbioavailability. By lowering corneal exposure, protecting cornealtissues, and increasing posterior segment bioavailability, theformulation of the present application improves ocular tolerability andincreases the therapeutic index of the active agent.

SUMMARY

The present application relates to pharmaceutical formulations in theform of a solution and/or a suspension, which lower the exposure to afirst active agent in the anterior segment of the eye, for example theocular surface comprised of the cornea and conjunctiva, and protect theeye, for example, the anterior segment of the eye, through maintenanceof EGFR activity. The pharmaceutical formulations of the applicationincrease the bioavailability of the first active agent at the posteriorsegment of the eye, for example at the central choroid and/or thecentral retina, and protect and/or improve the health of the anteriorsegment of the eye.

The present application provides a formulation comprising a first activeagent, and optionally a second active agent, in the form of a solutionor a suspension, with superior characteristics compared to a compositionformed as a gel. The present application provides that the first activeagent and/or the second active agent can be formulated together as asolution and/or a suspension. Increased levels of the first active agentin the anterior segment of the eye limit ocular tolerability of topicaldrops containing the first active agent and may cause corneal epithelialdefects and erosions. The presence of the second active agent canprevent damage that may be caused by exposure to the first active agent,treat any damage that may be caused by exposure to the first activeagent, and/or improve the overall health of the ocular surface,specifically the corneal epithelium. The second active agent can beformulated together with the first active agent, or formulated as aseparate formulation that is administered in combination with theformulation comprising the first active agent.

The formulations of the present application reduce exposure of the firstactive agent at the anterior segment of the eye, such as corneal orconjunctival surface, protect from and/or repair damages to the anteriorsegment of the eye, such as the corneal or conjunctival surface, andmaintain adequate concentrations of a first active agent necessary tobind the relevant receptors at the target tissues and confer atherapeutic effect in the posterior segment of the eye, such as thechoroid and the retina.

The present application relates to formulations and methods useful fortreating pathological states that arise from or are exacerbated byocular angiogenesis, neovascularization, and/or vascular leakage, forexample, in diabetic retinopathy (including background diabeticretinopathy, proliferative diabetic retinopathy, and diabetic macularedema); age-related macular degeneration (AMD) (including neovascular(wet/exudative) AMD, dry AMD, and Geographic Atrophy); pathologicchoroidal neovascularization (CNV) and vascular leakage from anymechanism (e.g., high myopia, trauma, sickle cell disease; ocularhistoplasmosis, angioid streaks, traumatic choroidal rupture, drusen ofthe optic nerve, or some retinal dystrophies); pathologic retinalneovascularization and vascular leakage from any mechanism (e.g., sicklecell retinopathy, Eales disease, ocular ischemic syndrome, carotidcavernous fistula, familial exudative vitreoretinopathy, hyperviscositysyndrome, idiopathic occlusive arteriolitis, birdshotretinochoroidopathy, retinal vasculitis, sarcoidosis, or toxoplasmosis);uveitis; retinal vein occlusion (central or branch); ocular trauma;surgery induced edema; surgery induced neovascularization; cystoidmacular edema; ocular ischemia; retinopathy of prematurity; Coat'sdisease; sickle cell retinopathy; and/or neovascular glaucoma. In oneembodiment, the pathological state is AMD. In one embodiment, thepathological states arise from or are exacerbated by ocular angiogenesisand/or neovascularization.

The present application also relates to formulations and methods usefulfor preventing and/or treating corneal epithelium disruptions associatedwith a disease, including a systemic disease (e.g., cancer, diabetes,etc.) and an eye disease, or with a side effect from a locally orsystemically administered drug (e.g., anti-EGFR agents or EGFRinhibitors). The formulation of the present application has, at least,one anti-angiogenic agent, anti-inflammatory agent, or anti-vascularpermeability agent for use in treating angiogenic ocular disorders andan EGFR modulator (e.g., an agent that maintains the activity of oractivates EGFR) for use in preventing and/or treating corneal epitheliumdisruptions.

According to embodiments of the application, the first active agent isan anti-angiogenic kinase inhibitor and the second active agent is akinase modulator (e.g., activator). In one embodiment, the first activeagent inhibits a kinase that is different from the kinase that ismodulated (e.g., activity maintained or activated) by the second activeagent. Examples of some kinase inhibitors that can be used to bringabout beneficial therapeutic results include inhibitors of receptortyrosine kinases, for example, without being limiting, VEGFR, FGFR,Tie-2, and Ephrin kinase receptors. Examples of kinase modulators thatcan be used to bring about beneficial therapeutic results includeactivators of ErbB receptor tyrosine kinase, for example, without beinglimiting, EGFR1/ErbB1/HER1, ErbB2/HER2/Neu, ErbB3/HER3, and ErbB4/HER.In one embodiment, the ErbB receptor tyrosine kinase is EGFR1. In oneembodiment, the first active agent is a VEGFR inhibitor. In oneembodiment, the second active agent is an EGFR modulator (e.g.,activator). In a further embodiment, the second active agent isnicotinic acid, nicotinamide, or vitamin K, or a combination thereof. Ina further embodiment, the second active agent is nicotinic acid ornicotinamide. In another embodiment, the second active agent vitamin K.

In some embodiments, a second active agent of the present applicationreduces or alleviates the inhibition of EGFR at the anterior segment ofthe eye caused by high concentrations of a first active agent of thepresent application (e.g., transient high concentrations of the firstactive agent after administration of the first active agent to theanterior segment of the eye).

The embodiments of the present application provide an ophthalmicformulation for treating ocular neovascularization comprising a firstactive agent of Formula I:

or a pharmaceutically acceptable salt thereof; a second active agentwherein the second active agent is an EGFR modulator (e.g., activator),such as nicotinic acid, nicotinamide, or vitamin K, or a combinationthereof; and pharmaceutically acceptable excipients; the first activeagent or the pharmaceutically acceptable salt is present in about 0.02%to about 1.2% w/v such that the formulation forms a solution orsuspension, and wherein:

X¹ is O or S;

R¹ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C(O)(C₁-C₁₀alkyl), (CH₂)_(t)(C₆-C₁₀ aryl), (CH₂)_(t) (4-10 membered heterocyclic),C(O)(CH₂)_(t)(C₆-C₁₀ aryl), or C(O)(CH₂)_(t) (5-10 memberedheterocyclic), wherein:

-   -   t is an integer from 0 to 5;    -   the alkyl group optionally includes 1 or 2 hetero moieties        selected from O, S and N(R⁶) with the proviso that two O atoms,        two S atoms, or an O and an S atoms are not attached directly to        each other;    -   the aryl and heterocyclic groups are optionally fused with a        C₆-C₁₀ aryl group, a C₅-C₈ saturated cyclic group, or a 5-10        membered heterocyclic group;    -   1 or 2 carbon atoms in the foregoing heterocyclic moieties are        optionally substituted with an oxo (═O) moiety or an anion of        oxygen;    -   the (CH₂)_(t) moieties optionally include a carbon-carbon double        or triple bond when t is an integer from 2 to 5; and    -   the foregoing R¹ groups, except H, are optionally substituted        with 1 to 3 R⁴ groups;

R² is H;

R³ is (CH₂)_(t)(C₆-C₁₀ aryl), wherein:

-   -   t is an integer from 0 to 5;    -   the aryl group is optionally fused with a C₆-C₁₀ aryl group, a        C₅-C₈ saturated cyclic group, or a 5-10 membered heterocyclic        group;    -   the (CH₂)_(t) moieties optionally include a carbon-carbon double        or triple bond when t is an integer from 2 to 5; and    -   the foregoing R³ groups are optionally substituted with 1 to 5        R⁴ groups;

each R⁴ is independently selected from C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, halo, cyano, nitro, trifluoromethyl, trifluoromethoxy,azido, OR⁵, C(O)R⁵, C(O)OR⁵, NR⁶C(O)R⁵, NR⁶C(O)OR⁵, OC(O)R⁵, NR⁶SO₂R⁵,SO₂NR⁵R⁶, C(O)NR⁵R⁶, NR⁵R⁶, S(O)_(j)R⁷ where j is an integer from 0 to2, SO₃H, NR⁵(CR⁶R⁷)_(t)OR⁶, (CH₂)_(t)(C₆-C₁₀ aryl), SO₂(CH₂)_(t)(C₆-C₁₀aryl), S(CH₂)_(t)(C₆-C₁₀ aryl), O(CH₂)_(t)(C₆-C₁₀ aryl), (CH₂)_(t) (5-10membered heterocyclic), and (CR⁶R⁷)_(m)OR⁶, wherein:

-   -   m is an integer from 1 to 5;    -   t is an integer from 0 to 5;    -   the alkyl group optionally includes 1 or 2 hetero moieties        selected from O, S and N(R⁶) with the proviso that two O atoms,        two S atoms, or an O and an S atoms are not attached directly to        each other;    -   the aryl and heterocyclic groups are optionally fused with a        C₆-C₁₀ aryl group, a C₅-C₈ saturated cyclic group, or a 5-10        membered heterocyclic group;    -   1 or 2 carbon atoms in the foregoing heterocyclic moieties are        optionally substituted with an oxo (═O) moiety or an anion of        oxygen; and    -   the alkyl, aryl and heterocyclic moieties of the foregoing R⁴        groups are optionally substituted with 1 to 3 substituents        independently selected from halo, cyano, nitro, trifluoromethyl,        trifluoromethoxy, azido, NR⁶SO₂R⁵, SO₂NR⁵R⁶, C(O)R⁵, C(O)OR⁵,        OC(O)R⁵, NR⁶C(O)R⁵, C(O)NR⁵R⁶, NR⁵R⁶, (CR⁶R⁷)_(m)OR⁶ where m is        an integer from 1 to 5, OR⁵, and the substituents listed in the        definition of R⁵; and

R⁵, R⁶, and R⁷ are each independently H or C₁-C₆ alkyl.

In one embodiment, R³ is (CH₂)_(t)(C₆-C₁₀ aryl), wherein t is an integerfrom 1 to 3 and R³ is optionally substituted with 1 to 4 R⁴ groups.

In a further embodiment, R³ is benzyl, optionally substituted with 1 to4 substituents independently selected from halo and C₁-C₄ alkyl. In afurther embodiment, R³ is benzyl substituted with 1 to 4 substituentsindependently selected from methyl, fluoro, chloro and bromo.

In one embodiment, R¹ is (CH₂)_(t) (5-10 membered heterocyclic), whereint is an integer from 0 to 5, optionally substituted with 1 or 2substituents independently selected from C₁-C₄ alkyl, hydroxy andhydroxymethyl.

The present application provides heterocyclic moiety of the R¹ group inFormula I selected from morpholino, pyrrolidinyl, imidazolyl,piperazinyl, piperidinyl, and 2,5-diaza-bicyclo[2.2.1]hept-2-yl, the tvariable of the R¹ group ranges from 2 to 5, and the R¹ group isoptionally substituted with one or more hydroxy groups.

For example, the heterocyclic moiety of the R¹ group in Formula I of thepresent application is pyrrolidine.

In a further embodiment of the present application, the first activeagent is:

In a further embodiment of the present application, the first activeagent is a hydrochloride salt of compound of Formula II, namelyCompound-I:

In one embodiment, the second active agent is an EGFR modulator (e.g.,activator). In a further embodiment, the second active agent isnicotinic acid, nicotinamide, or vitamin K, or a combination thereof. Ina further embodiment, the second active agent is vitamin K.

The embodiments of the present application provide formulationscomprising about 0.005% to about 5.0% w/v of the first active agent ofFormula I or II, or a pharmaceutically acceptable salt thereof, forexample, Compound-I. In some embodiments, the concentration ofCompound-I or its free base (Formula II) in the formulations is about0.005%-about 0.01%, about 0.01%-about 0.05%, about 0.05%-about 0.1%,about 0.1%-about 0.2%, about 0.2%-about 0.3%, about 0.3%-about 0.4%,about 0.4%-about 0.5%, about 0.5%-about 0.6%, about 0.6%-about 0.7%,about 0.7%-about 0.8%, about 0.8%-about 0.9%, about 0.9%-about 1.0%,about 1.1-about 2.0%, about 2.1-about 3.0%, about 3.1-about 4.0%, orabout 4.1-about 5.0% w/v for topical administration. In someembodiments, the concentration of Compound-I or its free base (FormulaII) in the formulations is about 0.1%-about 1.2%, about 0.2%-about 1.2%,about 0.3%-about 1.2%, about 0.4%-about 1.2%, 0.1%-about 1.1%, about0.2%-about 1.1%, about 0.3%-about 1.1%, about 0.4%-about 1.1%,0.1%-about 1.0%, about 0.2%-about 1.0%, about 0.3%-about 1.0%, about0.4%-about 1.0%, 0.1%-about 0.8%, about 0.2%-about 0.8%, about0.3%-about 0.8%, about 0.4%-about 0.8%, 0.1%-about 0.6%, about0.2%-about 0.6%, about 0.3%-about 0.6%, about 0.4%-about 0.6%,0.1%-about 0.5%, about 0.2%-about 0.5%, about 0.3%-about 0.5%, about0.4%-about 0.5%, 0.1%-about 0.4%, about 0.2%-about 0.4%, about0.3%-about 0.4% w/v for topical administration. In some embodiments theformulations include about 0.005%, about 0.05%, about 0.1%, about 0.2%,about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%,about 0.9%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, or about5.0% w/v of Compound-I or its free base (Formula II).

In some embodiments, the present application provides a solution of afirst active agent (e.g., Compound-I) and a second active agent (e.g.,nicotinic acid, nicotinamide, or vitamin K, or a combination thereof),which includes one or more solubilizing agents.

In some embodiments, the formulation comprises about 0.005%, about0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 2.0%, about3.0%, about 4.0%, or about 5.0% w/v of Formula I or II, or apharmaceutically acceptable salt thereof, for example, Compound-I, and asecond active agent and a solubilizing agent.

In some embodiments, the solubilizing agent in the formulation may becyclodextrin, for example, 2-hydroxypropyl-β-cyclodextrin,methyl-β-cyclodextrin, randomly methylated-β-cyclodextrin,ethylated-β-cyclodextrin, triacetyl-β-cyclodextrin,peracetylated-β-cyclodextrin, carboxymethyl-β-cyclodextrin,hydroxyethyl-β-cyclodextrin,2-hydroxy-3-(trimethylammonio)propyl-β-cyclodextrin,glucosyl-β-cyclodextrin, maltosyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, branched-β-cyclodextrin,hydroxypropyl-γ-cyclodextrin, randomly methylated-γ-cyclodextrin,trimethyl-γ-cyclodextrin, or a combination thereof.

In one embodiment, the solubilizing agent in the formulation is2-hydroxypropyl-β-cyclodextrin or β-cyclodextrin sulfobutyl ether.

In one embodiment, the formulation may further comprise one or more ofbenzalkonium chloride (BAK), sodium chloride, and a pH adjusting agent.

In additional embodiments, the formulation comprises about 0.005%, about0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 2.0%, about3.0%, about 4.0%, or about 5.0% w/v of a first active agent or apharmaceutically acceptable salt thereof, and a buffer, for example,tromethamine. In one embodiment, the formulation comprises about 0.005%,about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%,about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 2.0%,about 3.0%, about 4.0%, or about 5.0% w/v of the first active agent or apharmaceutically acceptable salt thereof, and about 0.3%-about 1.0% w/vtromethamine, and optionally further comprises about 0.005% w/vbenzalkonium chloride (BAK).

In some embodiments, the second active agent is an EGFR modulator (e.g.,activator). For example, the second active agent includes, but is notlimited to, nicotinic acid (or niacin/vitamin B3), nicotinamide, andvitamin K, or a combination thereof. “Vitamin K,” as defined herein,includes one or more members of the vitamin K family and prodrugsthereof, naturally occurring or synthetic. The vitamin K family iscomprised of vitamin K₁ (also known as phylloquinone, phytomenadione orphytonadione), vitamin K₂, and any vitamin K₂ homologues. The vitamin K₂homologues are called menoquinones and are characterized by the numberof isoprenoid residues in their sidechains. Synthetic vitamin Kincludes, but is not limited to vitamin K₃ (i.e., menadione), vitaminK₄, vitamin K₅. In one embodiment, the second active agent is nicotinicacid and/or and nicotinamide. In another embodiment, the second activeagent is nicotinic acid. In another embodiment, the second active agentis nicotinamide. In yet another embodiment, the second active agent isvitamin K. In another embodiment, the second active agent is vitamin K₁.In yet another embodiment, the second active agent is vitamin K₂. In yetanother embodiment, the second active agent is a vitamin K₂ homologue.In yet another embodiment, the second active agent is a syntheticvitamin K (e.g., vitamin K₃, vitamin K₄, or vitamin K₅). In oneembodiment, the second active agent is vitamin K₃ (i.e., menadione).

In some embodiments, the concentration of the second active agent in theformulations is about 0.00001%-about 5.0% w/v for topicaladministration. In some embodiments, the concentration of the secondactive agent in the formulations is about 0.00001%-about 1.0%, about0.00001%-about 0.1%, about 0.00001%-about 0.01%, about 0.00001%-about0.001%, about 0.00001%-about 0.0002%, or about 0.00001%-about 0.0001%w/v for topical administration. In some embodiments, the concentrationof the second active agent in the formulations is about 0.00001%-about0.0001%, 0.000012%-about 0.0001%, 0.000014%-about 0.0001%,0.000016%-about 0.0001%, 0.000018%-about 0.0001%, 0.00002%-about0.0001%, 0.00003%-about 0.0001%, 0.00004%-about 0.0001%, 0.00005%-about0.0001%, 0.00006%-about 0.0001%, 0.00007%-about 0.0001%, 0.00008%-about0.0001%, 0.00009%-about 0.0001%, 0.000016%-about 0.00009%,0.000018%-about 0.00009%, 0.00002%-about 0.00009%, 0.00003%-about0.00009%, 0.00004%-about 0.00009%, 0.00005%-about 0.00009%,0.00006%-about 0.00009%, 0.00007%-about 0.00009%, or 0.00008%-about0.00009% w/v for topical administration. In some embodiments, theformulations include about 0.00001%, 0.00002%, 0.00003%, 0.00004%,0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.000081%, 0.000082%, 0.000083%,0.000084%, 0.000085%, 0.000086%, 0.000087%, 0.000088%, or 0.000089% w/vof the second active agent, or a pharmaceutically acceptable saltthereof.

In some embodiments, the concentration of the second active agent in theformulations is about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM,about 0.9 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5μM, about 6 μM, about 7 μM, about 8 μM, or about 9 μM. In someembodiments, the concentration of the second active agent is about 1 μM.

The present application provides a formulation having a pH value ofabout 4.5 to about 7.5 at or under about 40° C. In some embodiments, thepH value of the formulation is between about pH 5.0 to about 7.0. In oneembodiment, the pH value of the formulation is about 6.0 at or underabout 40° C.

In another embodiment, the present application provides use of aformulation comprising a first active agent (e.g., Formula I orCompound-I or its free base (Formula II)), and a second active agent(e.g., nicotinic acid, nicotinamide, or vitamin K, or a combinationthereof), for the manufacture of a medicament for accessing posteriorsegment of the eye and/or for treating and/or ameliorating a posteriorsegment disease or a vasculopathic or inflammatory disease of the eye,as described herein, e.g., diabetic retinopathy (including backgrounddiabetic retinopathy, proliferative diabetic retinopathy and diabeticmacular edema); age-related macular degeneration (AMD) (includingneovascular (wet/exudative) AMD, dry AMD, and Geographic Atrophy);pathologic choroidal neovascularization (CNV) from any mechanism (e.g.,high myopia, trauma, sickle cell disease; ocular histoplasmosis, angioidstreaks, traumatic choroidal rupture, drusen of the optic nerve, andsome retinal dystrophies); pathologic retinal neovascularization fromany mechanism (e.g., sickle cell retinopathy, Eales disease, ocularischemic syndrome, carotid cavernous fistula, familial exudativevitreoretinopathy, hyperviscosity syndrome, idiopathic occlusivearteriolitis; birdshot retinochoroidopathy, retinal vasculitis,sarcoidosis and toxoplasmosis); uveitis; retinal vein occlusion (centralor branch); ocular trauma; surgery induced edema; surgery inducedneovascularization; cystoid macular edema; ocular ischemia; retinopathyof prematurity; Coat's disease; sickle cell retinopathy and/orneovascular glaucoma. In one embodiment, the disease of the eye is AMD.In one embodiment, the diseases of the eye arise from or are exacerbatedby ocular angiogenesis and/or neovascularization.

In another embodiment, the present application relates to a formulationfor use in the manufacture of a medicament suitable for accessing theposterior segment of the eye and/or for treating and/or ameliorating aposterior segment disease or a vasculopathic or inflammatory disease ofthe eye. In one embodiment, the formulation comprises a first activeagent and a second active agent. In one embodiment, the first activeagent is a VEGFR inhibitor. In one embodiment, the first active agent isa compound of Formula I or a pharmaceutically acceptable salt thereof.In a further embodiment, the first active agent is a compound of FormulaII or a pharmaceutically acceptable salt thereof. In a furtherembodiment, the first active agent is Compound-I. In one embodiment, thesecond active agent is an EGFR modulator (e.g., activator). In a furtherembodiment, the second active agent is selected from nicotinic acid,nicotinamide, and vitamin K, and a combination thereof. The formulationmay further comprise one or more pharmaceutically acceptable excipients.

In another embodiment, the present application relates to a formulationfor use in the manufacture of a medicament for treating and/orameliorating a symptom of an ocular disease or disorder (e.g., aposterior segment or vasculopathic or inflammatory disease of the eye).In one embodiment, the formulation comprises a first active agent and asecond active agent. In one embodiment, the first active agent is aVEGFR inhibitor. In one embodiment, the first active agent is a compoundof Formula I or a pharmaceutically acceptable salt thereof. In a furtherembodiment, the first active agent is a compound of Formula II or apharmaceutically acceptable salt thereof. In a further embodiment, thefirst active agent is Compound-I. In one embodiment, the second activeagent is an EGFR modulator (e.g., activator). In a further embodiment,the second active agent is selected from nicotinic acid, nicotinamide,and vitamin K, and a combination thereof. The formulation may furthercomprise one or more pharmaceutically acceptable excipients.

In yet another embodiment, the present application relates to aformulation for use in a method for accessing posterior segment of theeye and/or for treating and/or ameliorating a posterior segment diseaseof the eye. In one embodiment, the formulation comprises a first activeagent and a second active agent. In one embodiment, the first activeagent is a VEGFR inhibitor. In one embodiment, the first active agent isa compound of Formula I or a pharmaceutically acceptable salt thereof.In a further embodiment, the first active agent is a compound of FormulaII or a pharmaceutically acceptable salt thereof. In a furtherembodiment, the first active agent is Compound-I. In one embodiment, thesecond active agent is an EGFR modulator (e.g., activator). In oneembodiment, the second active agent is selected from nicotinic acid,nicotinamide, and vitamin K, and a combination thereof. The formulationmay further comprise one or more pharmaceutically acceptable excipients.

In yet another embodiment, the present application relates to aformulation for use in a method for treating and/or ameliorating asymptom of an ocular disease or disorder (e.g., a posterior segmentvasculopathic or inflammatory disease of the eye). In one embodiment,the formulation comprises a first active agent and a second activeagent. In one embodiment, the first active agent is a VEGFR inhibitor.In one embodiment, the first active agent is a compound of Formula I ora pharmaceutically acceptable salt thereof. In a further embodiment, thefirst active agent is a compound of Formula II or a pharmaceuticallyacceptable salt thereof. In a further embodiment, the first active agentis Compound-I. In one embodiment, the second active agent is an EGFRmodulator (e.g., activator). In a further embodiment, the second activeagent is selected from nicotinic acid, nicotinamide, and vitamin K, anda combination thereof. The formulation may further comprise one or morepharmaceutically acceptable excipients.

In another embodiment, the present application relates to acombinational therapy for accessing the posterior segment of the eyeand/or for treating and/or ameliorating a posterior segment disease ofthe eye, wherein the therapy comprises administering a first activeagent and a second active agent. In one embodiment, the first activeagent is a VEGFR inhibitor. In one embodiment, the first active agent isa compound of Formula I or a pharmaceutically acceptable salt thereof.In a further embodiment, the first active agent is a compound of FormulaII or a pharmaceutically acceptable salt thereof. In a furtherembodiment, the first active agent is Compound-I. In one embodiment, thesecond active agent is an EGFR modulator (e.g., activator). In a furtherembodiment, the second active agent is selected from nicotinic acid,nicotinamide, and vitamin K, and a combination thereof. In oneembodiment, the first active agent is administered simultaneously withthe second active agent. In another embodiment, the first active agentis administered prior to the administration of the second active agent.In another embodiment, the first active agent is administered after theadministration of the second active agent.

In another embodiment, the present application relates to acombinational therapy for treating and/or ameliorating a symptom of anocular disease or disorder (e.g., a posterior segment vasculopathic orinflammatory disease of the eye), comprising administering a firstactive agent and a second active agent. In one embodiment, the firstactive agent is a VEGFR inhibitor. In one embodiment, the first activeagent is a compound of Formula I or a pharmaceutically acceptable saltthereof. In a further embodiment, the first active agent is a compoundof Formula II or a pharmaceutically acceptable salt thereof. In afurther embodiment, the first active agent is Compound-I. In oneembodiment, the second active agent is an EGFR modulator (e.g.,activator). In a further embodiment, the second active agent is selectedfrom nicotinic acid, nicotinamide, and vitamin K, and a combinationthereof. In one embodiment, the first active agent is administeredsimultaneously with the second active agent. In another embodiment, thefirst active agent is administered prior to the administration of thesecond active agent. In another embodiment, the first active agent isadministered after the administration of the second active agent.

In some embodiments, the exposure time of the first active agent (e.g.,Compound-I) and the second active agent is between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments, the exposure time of the first active agent (e.g.,Compound-I) and the second active agent is longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments, thedosage regimen involves several courses of topical ocular administrationof a formulation comprising the first active agent (e.g., Compound-I)and a second active agent to a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months)or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or 12months) or for longer than 90 days. For example, the dosage regimeninvolves once daily, twice daily, three times daily or four times dailyadministration of the formulation for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months)or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or 12months). For example, the dosage regimen involves once, twice, threetimes, or four times administration of the formulation on alternate days(i.e., on day 1, 3, 5, 7 etc.) for up to 90 days. For example, thedosage regimen involves administering once on day 1, once or twice onday 2 to day 90. For example, the dosage regimen involves administeringonce, twice, three times, or four times on day 1, followed by once dailyfor 2-90 days. For example, the dosage regimen involves administeringonce, twice, three times, four times on day 1, followed by once, twice,three times, or four times on alternate days (i.e., on day 1, 3, 5, 7etc.) for up to 90 days. For example, one dosage regimen involvesadministering once or twice per day for 1, 2, 3, 4, or 5 consecutivedays. For twice or three daily dosage regimen, subjects receive atopical ocular dose of a first active agent (e.g., Compound-I) and asecond active agent formulation on days 1 and 4 approximately about 4,6, or 8 hours apart. In another embodiment, subjects receive topicalocular doses of a first active agent (e.g., Compound-I) and a secondactive agent formulation approximately about 4, 6, or 8 hours apart forfour consecutive days. In some embodiments, subjects receive one or twotopical ocular doses of a first active agent (e.g., Compound-I) and asecond active agent formulation per day for 5 consecutive days. In yetother embodiments, subjects receive one or two topical ocular dose of afirst active agent (e.g., Compound-I) and a second active agentformulation for 5-90 consecutive days. In some embodiments, subjectsreceive one or two topical ocular doses of a first active agent (e.g.,Compound-I) and a second active agent formulation for at least 25consecutive days. In one embodiment, subjects receive one or two topicalocular doses for at least 90 consecutive days or more.

For example, a formulation comprising about 1 mg/mL BID of a firstactive agent (e.g., Compound-I) and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months) or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments a formulation comprisingabout 1 mg/mL QD of a first active agent (e.g., Compound-I) and a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 1 mg/mL TID of a first active agent (e.g., Compound-I)and a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 1 mg/mL QID of a first active agent (e.g.,Compound-I) and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodimentsa formulation comprising about 2 mg/mL BID of a first active agent(e.g., Compound-I) and a second active agent is administered to one eyeor both eyes of a subject for between 1 and 90 days or for longer than90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 2 mg/mL QD of a first activeagent (e.g., Compound-I) and a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 2 mg/mL TID of afirst active agent (e.g., Compound-I) and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments a formulation comprising about 2 mg/mLQID of a first active agent (e.g., Compound-I) and a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 3mg/mL BID of a first active agent (e.g., Compound-I) and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments a formulation comprisingabout 3 mg/mL QD of a first active agent (e.g., Compound-I) and a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 3 mg/mL TID of a first active agent (e.g., Compound-I)and a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 3 mg/mL QID of a first active agent (e.g.,Compound-I) and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodimentsa formulation comprising about 4 mg/mL BID of a first active agent(e.g., Compound-I) and a second active agent is administered to one eyeor both eyes of a subject for between 1 and 90 days or for longer than90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 4 mg/mL QD of a first activeagent (e.g., Compound-I) and a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 4 mg/mL TID of afirst active agent (e.g., Compound-I) and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments a formulation comprising about 4 mg/mLQID of a first active agent (e.g., Compound-I) and a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 5mg/mL BID of a first active agent (e.g., Compound-I) and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments a formulation comprisingabout 5 mg/mL QD of a first active agent (e.g., Compound-I) and a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 5 mg/mL TID of a first active agent (e.g., Compound-I)and a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 5 mg/mL QID of a first active agent (e.g.,Compound-I) and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodimentsa formulation comprising about 6 mg/mL BID of a first active agent(e.g., Compound-I) and a second active agent is administered to one eyeor both eyes of a subject for between 1 and 90 days or for longer than90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 6 mg/mL QD of a first activeagent (e.g., Compound-I) and a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 6 mg/mL TID of afirst active agent (e.g., Compound-I) and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments a formulation comprising about 6 mg/mLQID of a first active agent (e.g., Compound-I) and a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 7mg/mL BID of a first active agent (e.g., Compound-I) and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments a formulation comprisingabout 7 mg/mL QD of a first active agent (e.g., Compound-I) and a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 7 mg/mL TID of a first active agent (e.g., Compound-I)and a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 7 mg/mL QID of a first active agent (e.g.,Compound-I) and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodimentsa formulation comprising about 8 mg/mL BID of a first active agent(e.g., Compound-I) and a second active agent is administered to one eyeor both eyes of a subject for between 1 and 90 days or for longer than90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 8 mg/mL QD of a first activeagent (e.g., Compound-I) and a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 8 mg/mL TID of afirst active agent (e.g., Compound-I) and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments a formulation comprising about 8 mg/mLQID of a first active agent (e.g., Compound-I) and a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 9mg/mL BID of a first active agent (e.g., Compound-I) and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments a formulation comprisingabout 9 mg/mL QD of a first active agent (e.g., Compound-I) and a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 9 mg/mL TID of a first active agent (e.g., Compound-I)and a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 9 mg/mL QID of a first active agent (e.g.,Compound-I) and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodimentsa formulation comprising about 10 mg/mL QD of a first active agent(e.g., Compound-I) and a second active agent is administered to one eyeor both eyes of a subject for between 1 and 90 days or for longer than90 days (e.g., 4 months, 6 months, 8 months, or 12 months). The dosageregimen for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months) may be any of the regimensinvolving consecutive or alternate days described in the paragraphabove. In some embodiments, the formulation of the present applicationis administered QD or BID. In some embodiments, the formulation of thepresent application is administered QD, BID, TID, or QID whenadministered at low doses (e.g., 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, or5 mg/mL), and QD or BID at high doses (e.g., 6 mg/mL, 7 mg/mL, 8 mg/mL,9 mg/mL, or 10 mg/mL).

In some embodiments, the formulation of a first active agent (e.g.,Formula II or Compound-I) and a second active agent is administered toone eye or both eyes of a subject. For example, about 0.2%-about 1.0%(w/v) of the compound of Formula II or about 0.1%-1.2% (w/v) ofCompound-I and a second active agent comprising formulation of thepresent application is administered once a day (QD), twice a day (BID),three times a day (TID), or four times a day (QID) to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodiments,Formula II compound or Compound-I is complexed with a complexing agent,e.g., cyclodextrin (e.g., hydroxypropyl-β-cyclodextrin (HP-β-CD,KLEPTOSE® HPB) (%)) in ratio of about 1:8, in which about 2%-13% (w/v)cyclodextrin (e.g., KLEPTOSE® HPB (%)) is added to the formulation. Theformulation may further comprise about 0.1%-about 0.2% buffer, e.g., 10mM phosphate buffer. The desired osmolality of the formulation may beabout 200-about 300 mOsm, achieved by adding quantity sufficient toachieve the osmolality with a salt, e.g., sodium chloride. The pH of theformulation may be about 6.0.

The present application relates to a pharmaceutical compositioncomprising particles of an active agent of the present application(e.g., a first active agent (e.g., Formula II or Compound-I) and/or asecond active agent), or a pharmaceutically acceptable salt thereof,wherein the particles have a mean diameter of between 100 nm and 100 μm.

The present application relates to a suspension formulation comprising apharmaceutical composition, wherein the pharmaceutical compositioncomprises particles of an active agent of the present application (e.g.,a first active agent (e.g., Formula II or Compound-I) and/or a secondactive agent), or a pharmaceutically acceptable salt thereof, asdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of immunoblots of EGFR phosphorylation inimmortalized corneal epithelial cells (hTCEpi cells) treated withvarying concentrations of a first active agent of the presentapplication or a control (AG1478, an EGFR kinase inhibitor) and of EGF(top panels: immunoblotting of total EGFR, middle panels: immunoblottingshowing phosphorylation of tyrosine 1068 of EGFR, bottom panels:immunoblotting showing phosphorylation of tyrosine 1045 of EGFR).

FIG. 2 is a series of immunoblots of EGFR phosphorylation inimmortalized corneal epithelial cells (hTCEpi cells) treated withvarying concentrations of a first active agent of the presentapplication either alone, or after treatment with vitamin K₃ (50 μM),nicotinic acid (10 μM), or nicotinamide (10 μM) (first row:immunoblotting of total EGFR, second row: immunoblotting showingphosphorylation of tyrosine 1068 of EGFR, third row: immunoblottingshowing phosphorylation of tyrosine 1045 of EGFR).

FIG. 3A is a series of micrographs showing the migration/proliferationof immortalized corneal epithelial cells (hTCEpi cells) treated for 30minutes with the indicated concentrations of a first active agent of thepresent application or a control (AG1478, an EGFR kinase inhibitor),followed by 16 hour treatment with the first active agent or controltogether with the indicated concentrations of EGF or VEGF. FIG. 3B is aseries of bar graphs quantifying the migration/proliferation of hTCEpicells in FIG. 3A.

FIG. 4A is a series of micrographs showing the migration/proliferationof immortalized corneal epithelial cells (hTCEpi cells) treated with theindicated concentrations of vitamin K₃, menadione, for 4 hours, and thensupplemented with varying concentrations of a first active agent of thepresent application, followed by treatment with vitamin K₃, the firstactive agent together with the EGF. FIG. 4B is a series of bar graphsquantifying the migration/proliferation of hTCEpi cells in FIG. 4A.

FIG. 5A is a series of images of epithelial wounds at the time of theinitial wounding (0 hr) and post-wounding of corneas treated withvehicle, the indicated concentrations of a compound of Formula I or II,or AG1478. FIGS. 5B and 5C are a series of bar graphs quantifying thewound healing at 16 hours (FIG. 5B) or 24 hours (FIG. 5C) post woundingof corneas treated with vehicle, the indicated concentrations of acompound of Formula I or II, or AG1478. FIG. 5D is a graph showing thetime-course wound healing of corneas treated with vehicle, the indicatedconcentrations of a compound of Formula I or II, or AG1478.

FIG. 6A is a series of images of epithelial wounds at the time of theinitial wounding (0 hr) and post-wounding of corneas treated withvehicle, menadione, a compound of Formula I or II, or menadione incombination with a compound of Formula I or II. FIG. 6B is a series ofbar graphs quantifying the wound healing at 16 hours or 24 hours postwounding of corneas treated with vehicle, menadione, a compound ofFormula I or II, or menadione in combination with a compound of FormulaI or II. FIG. 6C is a graph showing the time-course wound healing ofcorneas treated with vehicle, menadione, a compound of Formula I or II,or menadione in combination with a compound of Formula I or II.

FIG. 7A is a series of immunoblots showing the total VEGFR2,phosphorylated VEGFR2, and α-tubulin (as loading control) in humanretinal endothelial cells treated with the indicated concentrations ofmenadione for 4 hours, followed by 30 minutes treatment with varyingconcentrations of a compound of Formula I or II (1 nM, 10 nM, 100 nM, or1 μM), and then 10 ng/ml VEGF. FIG. 7B is a graph quantifying growth ofhuman retinal endothelial cells following treatment with the indicatedconcentrations of menadione for 4 hours, followed by 30 minutestreatment with varying concentrations of a compound of Formula I or II(0.1 nM, 1 nM, 10 nM, or 100 nM), and then 10 ng/ml VEGF overnight.

FIG. 8 is a series of immunoblots showing the total EGFR2,phosphorylated EGFR, and α-Tubulin (as loading control) in hTCEpi cellstreated with the indicated concentrations of menadione for 4 hours,followed by incubation with 8.0 nM EGF for the indicated periods oftime.

FIG. 9A shows the size distribution of the particles comprising 5% of acompound of Formula II and 1% Pluronic F-127 produced using smallmilling media for a long period of time at a high roller speed. FIG. 9Bshows the size distribution of the particles comprising 5% of a compoundof Formula II and 1% Pluronic F-127 produced using large milling mediafor a short period of time at a low roller speed. FIG. 9C shows the sizedistribution of the particles comprising 3% of Compound-I, 0.6% TrisHCl, and 2% glycerol produced without milling. FIG. 9D shows the sizedistribution of the particles comprising 0.4% of a compound of FormulaII, 10 μM menadione, 0.08% Pluronic F-127, and 2.5% glycerol afterstorage at 40° C. for 7 days.

FIG. 10 shows the size distribution of the particles comprising 5% ofmenadione and 1% HPMC.

FIGS. 11A and 11B show the size distribution of the particles comprising5% of a compound of Formula II and 1% Pluronic F-127 produced usinglarge milling media for a short period of time at a low roller speed.

DETAILED DESCRIPTION

The materials, compounds, compositions, articles, and methods describedherein may be understood more readily by reference to the followingdetailed description of specific aspects of the disclosed subject matterand the Examples included therein. Before the present materials,compounds, compositions, articles, devices, and methods are disclosedand described, it is to be understood that the aspects described beloware not limited to specific methods or specific reagents, as such mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular aspects only and is notintended to be limiting.

Also, throughout this specification, various publications arereferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which the disclosed matterpertains. The references disclosed are also individually andspecifically incorporated by reference herein for the material containedin them that is discussed in the sentence in which the reference isrelied upon.

The present application provides compositions or formulations thatcontain a first active agent and/or a second active agent for use in thetreatment of ocular disorders caused by endothelial cell proliferation,enhanced vascular permeability, inflammation, angiogenesis, orneovascularization, and a second active agent for use in the preventionand/or treatment of damage caused to the anterior of the eye by thefirst active agent, a systemic disease and/or an eye disease.

The present application also relates to a combination of a first activeagent, e.g., a compound of Formula I or II, and a second active agent,e.g., nicotinic acid, nicotinamide, vitamin K, or a combination thereof.In one embodiment, a compound of Formula I or II, or a pharmaceuticallyacceptable salt thereof, and a second active agent or a pharmaceuticallyacceptable salt thereof, are administered simultaneously. Alternatively,a compound of Formula I or II, or a pharmaceutically acceptable saltthereof, is administered prior to administration of a second activeagent, or a pharmaceutically acceptable salt thereof. In anotherembodiment, a compound of Formula I or II, or a pharmaceuticallyacceptable salt thereof, is administered after administration of asecond active agent, or a pharmaceutically acceptable salt thereof.

The formulations of the application are useful in preventing orinhibiting neovascularization and vascular leakage associated withocular disorders while preventing or inhibiting corneal diseases. Insome cases, the formulations of the application cause regression ofneovascularization. Briefly, within the context of the presentapplication, the first active agents should be understood to be anymolecule, either synthetic or naturally occurring, which acts to inhibitvascular growth, reduce vascular permeability, and/or decreaseinflammation.

The formulations of the application are also useful in preventing and/ortreating corneal epithelium disruptions caused by systemic diseases(e.g., cancer, diabetes, etc.), eye diseases, or a side effect from alocally or systemically administered drug (e.g., anti-EGFR agents, orcompounds of Formula I or II having anti-EGFR activity). Briefly, withinthe context of the present application, the second active agents shouldbe understood to be any molecule, either synthetic or naturallyoccurring, which acts to protect from and/or repair corneal edema,ulceration or any other corneal abnormality. In particular, the presentapplication provides formulations comprising a first active agent and asecond active agent each in a therapeutically effective amount.

General Definitions

In this specification and in the claims that follow, reference is madeto a number of terms, which shall be defined to have the followingmeanings. All percentages, ratios and proportions herein are by weight,unless otherwise specified. All temperatures are in degrees Celsius (°C.) unless otherwise specified.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material can beadministered to an individual along with the relevant active compoundwithout causing clinically unacceptable biological effects orinteracting in a deleterious manner with any of the other components ofthe pharmaceutical composition or formulation in which it is contained.

A weight percent of a component, unless specifically stated to thecontrary, is based on the total weight of the formulation or compositionin which the component is included.

By “effective amount” as used herein means “an amount of one or more ofthe disclosed compounds, effective at dosages and for periods of timenecessary to achieve the desired or therapeutic result.” An effectiveamount may vary according to factors known in the art, such as thedisease state, age, sex, and weight of the human or animal beingtreated. Although particular dosage regimes may be described in examplesherein, a person skilled in the art would appreciate that the dosageregime may be altered to provide optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation. In addition, the formulations of this disclosurecan be administered as frequently as necessary to achieve a therapeuticamount.

The effective amount or effective dose in a human can be determined fromthat in an animal (e.g., an experimental animal). For example, theeffective dose in a human may be calculated based on the conversionshown in the table below.

Conversion of animal doses to human-equivalent doses (HEDs) by using theexponent 0.67 for body surface area

HED from HED from mg · kg⁻¹ dose in mg · kg⁻¹ dose in animal divideanimal multiply Species animal dose by animal dose by Mouse 12.3 0.081Hamster 7.4 0.135 Rat 6.2 0.162 Ferret 5.3 0.189 Guinea pig 4.6 0.216Rabbit 3.1 0.324 Dog 1.8 0.541 Monkey 3.1 0.324 Marmoset 6.2 0.162Squirrel monkey 5.3 0.189 Baboon 1.8 0.541 Micro-pig 1.4 0.730 Mini-pig1.1 0.946

“Excipient” is used herein to include any other compound that may becontained in or combined with one or more of the disclosed inhibitorsthat is not a therapeutically or biologically active compound. As such,an excipient should be pharmaceutically or biologically acceptable orrelevant (for example, an excipient should generally be non-toxic to thesubject). “Excipient” includes a single such compound and is alsointended to include a plurality of excipients. For the purposes of thepresent application the term “excipient” and “carrier” are usedinterchangeably throughout the description of the present applicationand said terms are defined herein as, “ingredients which are used in thepractice of formulating a safe and effective pharmaceuticalcomposition.”

As used herein, by a “subject” is meant an individual. Thus, the“subject” can include domesticated animals (e.g., cats, dogs, etc.),livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratoryanimals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds.“Subject” can also include a primate or a human.

By “reduce” or other forms of the word, such as “reducing” or“reduction,” is meant lowering of an event or characteristic (e.g.,vascular leakage, or tissue swelling). It is understood that this istypically in relation to some standard or expected value, in other wordsit is relative, but that it is not always necessary for the standard orrelative value to be referred to.

The term “treat” or other forms of the word such as “treated” or“treatment” is used herein to mean that administration of a compound orformulation of the present application mitigates a disease or a disorderin a host and/or reduces, inhibits, or eliminates a particularcharacteristic or event associated with a disorder (e.g., vascularleakage or corneal ulceration).

Insofar as the methods of the present application are directed topreventing disorders, it is understood that the term “prevent” does notrequire that the disease state be completely thwarted. Rather, as usedherein, the term preventing refers to the ability of the skilled artisanto identify a population that is susceptible to disorders, such thatadministration of the compounds of the present application may occurprior to onset of a disease. The term does not imply that the diseasestate be completely avoided.

The term “ameliorating” or other forms of the word such as “ameliorate”is used herein to mean that administration of a therapeutic agent of thepresent application mitigates one or more symptoms of a disease or adisorder in a host and/or reduces, inhibits, or eliminates a particularsymptom associated with the disease or disorder prior to and/or postadministration of the therapeutic agent.

The disclosed first active agent compounds affect vascular leakage orpathological neovascularization by inhibiting a receptor tyrosinekinase. The disclosed second active agent compounds affect cornealepithelium disruptions by modulating (e.g., activating) a receptortyrosine kinase.

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other additives, or components.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “about” refers to any minimal alteration in the concentrationor amount of a therapeutic agent (e.g., a first active agent or a secondactive agent) that does not change the efficacy of the agent inpreparation of a formulation and in treatment of a disease or disorder.For example, without being limiting, the concentration of a therapeuticagent would be effective if the concentration is varied between 0.005%to 5.0% (e.g., ±0.0005%). The term “about” with respect to concentrationrange of the therapeutic/active agents of the present application, e.g.,first active agent or second active agent, also refers to any variationof a stated amount or range which would be an effective amount or range.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it is understood thatthe particular value forms another aspect. It is further understood thatthe endpoints of each of the ranges are significant both in relation tothe other endpoint, and independently of the other endpoint. It is alsounderstood that there are a number of values disclosed herein, and thateach value is also herein disclosed as “about” that particular value inaddition to the value itself. For example, if the value “10” isdisclosed, then “about 10” is also disclosed. It is also understood thatwhen a value is disclosed, then “less than or equal to” the value,“greater than or equal to the value,” and possible ranges between valuesare also disclosed, as appropriately understood by the skilled artisan.For example, if the value “10” is disclosed, then “less than or equal to10” as well as “greater than or equal to 10” is also disclosed. It isalso understood that throughout the application data are provided in anumber of different formats and that this data represent endpoints andstarting points and ranges for any combination of the data points. Forexample, if a particular data point “10” and a particular data point“15” are disclosed, it is understood that greater than, greater than orequal to, less than, less than or equal to, and equal to 10 and 15 areconsidered disclosed as well as between 10 and 15. It is also understoodthat each unit between two particular units are also disclosed. Forexample, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are alsodisclosed.

The term “halo”, as used herein, unless otherwise indicated, includesfluoro, chloro, bromo or iodo. Preferred halo groups are fluoro, chloroand bromo.

The term “alkyl,” as used herein, unless otherwise indicated, includesboth branched and straight-chain saturated aliphatic hydrocarbon groupshaving the specified number of carbon atoms. For example, C₁₋₆alkyl isintended to include C₁, C₂, C₃, C₄, C₅, and C₆ alkyl groups. Examples ofalkyl include, but are not limited to, methyl, ethyl, n-propyl,i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl, and n-hexyl. Incertain embodiments, a straight chain or branched chain alkyl has six orfewer carbon atoms in its backbone (e.g., C₁-C₆ for straight chain,C₃-C₆ for branched chain), and in another embodiment, a straight chainor branched chain alkyl has four or fewer carbon atoms. Likewise,cycloalkyls have from three to eight carbon atoms in their ringstructure, and in other embodiments, cycloalkyls have five or sixcarbons in the ring structure.

Alkyl can be substituted by replacing hydrogen on one or more carbons ofthe hydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkylamino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above and additional substituents such as alkyl, alkenyl, andalkynyl. An “alkylaryl” or an “aralkyl” moiety is an alkyl substitutedwith an aryl (e.g., phenylmethyl (benzyl)).

The term “alkenyl,” as used herein, unless otherwise indicated, includesunsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double bond. For example, the term “alkenyl” includes straight-chainalkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl,heptenyl, octenyl, nonenyl, decenyl), branched-chain alkenyl groups,cycloalkenyl (e.g., alicyclic) groups (e.g., cyclopropenyl,cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl oralkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenylsubstituted alkenyl groups. In certain embodiments, a straight chain orbranched chain alkenyl group has six or fewer carbon atoms in itsbackbone (e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain).Likewise, cycloalkenyl groups may have from three to eight carbon atomsin their ring structure, and in some embodiments, cycloalkenyl groupshave five or six carbons in the ring structure. The term “C₂-C₆”includes alkenyl groups containing two to six carbon atoms. The term“C₃-C₆” includes alkenyl groups containing three to six carbon atoms.Alkenyl can be substituted by replacing hydrogen on one or morehydrocarbon backbone carbon atoms. Such substituents can include, forexample, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “alkynyl”, as used herein, unless otherwise indicated, includesunsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but which contain at leastone triple bond. For example, “alkynyl” includes straight-chain alkynylgroups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl,octynyl, nonynyl, decynyl), branched-chain alkynyl groups, andcycloalkyl or cycloalkenyl substituted alkynyl groups. In certainembodiments, a straight chain or branched chain alkynyl group has six orfewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). The term “C₂-C₆” includes alkynyl groupscontaining two to six carbon atoms. The term “C₃-C₆” includes alkynylgroups containing three to six carbon atoms. Alkynyl can be substitutedby replacing hydrogen on one or more hydrocarbon backbone carbon atoms.Such substituents can include, for example, halogens, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkylamino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkoxy,” as used herein, unless otherwise indicated, includesO-alkyl groups wherein “alkyl” is as defined above.

The term “aryl,” as used herein, unless otherwise indicated, includes 5-and 6-membered “unconjugated”, or single-ring, aromatic groups, as wellas “conjugated”, or multicyclic, systems with at least one aromaticring. Examples of aryl groups include benzene, phenyl, and the like.Furthermore, the term “aryl” includes multicyclic aryl groups, e.g.,tricyclic, bicyclic, e.g., naphthalene.

Aryl groups having heteroatoms in the ring structure may be referred toas “aromatic heterocycles”, “aryl heterocycles”, “heterocycles,”“heteroaryls” or “heteroaromatics”.

The aryl or heteroaryl can be substituted at one or more ring positionswith such substituents as described above, as for example, halogen,hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino(including alkylamino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Arylgroups can also be fused or bridged with alicyclic or heterocyclicrings, which are not aromatic so as to form a multicyclic system (e.g.,tetralin, methylenedioxyphenyl).

The term “4-10 membered heterocyclic,” as used herein, unless otherwiseindicated, includes aromatic and non-aromatic heterocyclic groupscontaining one or more heteroatoms each selected from O, S and N,wherein each heterocyclic group has from 4-10 atoms in its ring system.Non-aromatic heterocyclic groups include groups having only 4 atoms intheir ring system, but aromatic heterocyclic groups must have at least 5atoms in their ring system. An example of a 4 membered heterocyclicgroup is azetidinyl (derived from azetidine). An example of a 5 memberedheterocyclic group is thiazolyl and an example of a 10 memberedheterocyclic group is quinolinyl.

Examples of non-aromatic heterocyclic groups are pyrrolidinyl,tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl,homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl andquinolizinyl.

Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl,pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl,thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl,quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl,furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl,benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, andfuropyridinyl.

The foregoing groups, as derived from the compounds listed above, may beC-attached or N-attached where such is possible. For instance, a groupderived from pyrrole may be pyrrol-1-yl (N-attached) or pyrrol-3-yl(C-attached). The “4-10 membered heterocyclic” moiety can besubstituted.

The phrase “pharmaceutically acceptable salt(s),” as used herein, unlessotherwise indicated, includes salts of acidic or basic groups which maybe present in the compounds of first active agents (e.g., Formula I orII) or second active agents. The compounds of Formula I or II and thesecond active agents that are basic in nature are capable of forming awide variety of salts with various inorganic and organic acids. Theacids that may be used to prepare pharmaceutically acceptable acidaddition salts of such basic compounds of Formula I or II and secondactive agents are those that form non-toxic acid addition salts, i.e.,salts containing pharmacologically acceptable anions, such as thehydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate,phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate,citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts.

Those compounds of first active agents (e.g., Formula I or II) andsecond active agents that are acidic in nature are capable of formingbase salts with various pharmacologically acceptable cations. Examplesof such salts include the alkali metal or alkaline earth metal salts andparticularly, the sodium and potassium salts.

In some embodiments, the salt is an acid addition salt, e.g. HCl salt.

Certain compounds of Formula I or II and certain second active agentsmay have asymmetric centers and therefore exist in differentenantiomeric forms. This application relates to the use of all opticalisomers and stereoisomers of the compounds of Formula I or II andmixtures thereof and of the second active agents and mixtures thereof.The compounds of Formula I or II and the second active agents may alsoexist as E/Z geometric isomers or tautomers. This application relates tothe use of all such geometric isomers and tautomers and mixturesthereof.

The subject application also includes isotopically-labeled compounds,and the pharmaceutically acceptable salts thereof, which are identicalto those recited in Formula I or II, but for the fact that one or moreatoms are replaced by an atom having an atomic mass or mass numberdifferent from the atomic mass or mass number usually found in nature.Examples of isotopes that can be incorporated into compounds of theapplication include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O,¹⁷O, ³⁵S, ¹⁸F, and ³⁶Cl, respectively. Compounds of the Formula I or II,conjugates thereof, and pharmaceutically acceptable salts of saidcompounds or of said conjugates which contain the aforementionedisotopes and/or other isotopes of other atoms are within the scope ofthis application. Certain isotopically-labeled compounds of the presentapplication (e.g., compounds of Formula I or II), for example those intowhich radioactive isotopes such as ³H and ¹⁴C are incorporated, areuseful in drug and/or substrate tissue distribution assays. Tritiated,i.e., ³H, and carbon-14, i.e., ¹⁴C isotopes are particularly preferredfor their ease of preparation and detectability. Further, substitutionwith heavier isotopes such as deuterium, i.e., ²H, can afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements and,hence, may be preferred in some circumstances. Isotopically labeledcompounds of Formula I or II of this application and esters or lipidconjugates thereof can generally be prepared by carrying out theprocedures disclosed in the Schemes and/or in the Examples andPreparations below, by substituting a readily available isotopicallylabeled reagent for a non-isotopically labeled reagent.

This application also encompasses pharmaceutical formulations containingderivatives of compounds of the Formula I or II, or pharmaceuticallyacceptable salts thereof and of the second active agents, orpharmaceutically acceptable salts thereof. Compounds of Formula I or II,or pharmaceutically acceptable salts thereof, and second active agents,or pharmaceutically acceptable salts thereof, having free amino, oramido groups can be converted into conjugated derivatives, wherein anamino acid residue, or a polypeptide chain of two or more (e.g., two,three or four) amino acid residues is covalently joined through an amideor ester bond to a free amino group of compounds of Formula I or II, orpharmaceutically acceptable salts thereof, or of second active agents orpharmaceutically acceptable salts thereof. The amino acid residuesinclude, but are not limited to, the 20 naturally occurring amino acidscommonly designated by three letter symbols and also includes4-hydroxyproline, hydroxylysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline homocysteine, homoserine, ornithine and methionine sulfone.

Additional types of derivatives are also encompassed. Amide and estermoieties may incorporate groups including but not limited to ether,amine and carboxylic acid functionalities. Free hydroxy groups may bederivatized using groups including but not limited to hemisuccinates,phosphate esters, dimethylaminoacetates, andphosphoryloxymethyloxycarbonyls, as outlined in D. Fleisher, et al.,ADVANCED DRUG DELIVERY REVIEWS (1996) 19, 115. Carbamate conjugates ofhydroxy and amino groups are also included, as are carbonate conjugatesand sulfate esters of hydroxy groups. Derivatization of hydroxy groupsas (acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group maybe an alkyl ester, optionally substituted with groups including but notlimited to ether, amine and carboxylic acid functionalities, or wherethe acyl group is an amino acid ester as described above, are alsoencompassed. Derivatives of this type are described in R. P. Robinson etal., J. MEDICINAL CHEMISTRY (1996) 39, 10.

The term “kinase” refers to any enzyme that catalyzes the addition ofphosphate groups to a protein residue; for example, serine and threoninekinases catalyze the addition of phosphate groups to serine andthreonine residues.

The terms “VEGFR kinase,” and “VEGFR,” refer to any of the vascularendothelial growth factor receptors.

The terms “VEGF signaling,” and “VEGF cascade” refer to both theupstream and downstream components of the VEGF signaling cascade.

The terms “ErbB kinase,” and “ErbB receptor,” refer to any member of theErbB family of receptor tyrosine kinases including EGFR (ErbB1 or HER1),HER2/c-neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4).

The terms “EGF signaling,” and “EGF cascade” refer to both the upstreamand downstream components of the EGF signaling cascade.

The term “pharmaceutically acceptable” refers to the fact that thecarrier, diluent or excipient must be compatible with the otheringredients of the formulation and not deleterious to the recipientthereof.

The terms “administration of a compound” or “administering a compound”refer to the act of providing a compound of the application orpharmaceutical formulation to the subject in need of treatment.

The term “vasculostasis” refers to the maintenance of the homeostaticvascular functioning leading to the normal physiologic functioning.

The term “vasculostatic agents” refers to agents that seek to addressconditions in which vasculostasis is compromised by preventing the lossof or restoring or maintaining vasculostasis.

In the present application “composition” and “formulation” are usedinterchangeably and refer to the conventional understanding, as known inthe art, of a composition or formulation.

The present application relates to an ophthalmic formulation. In someembodiments, the ophthalmic formulation of the present application is agel formulation or a semi-gel formulation, or both.

“Gel” according to the present application is a semi-solid dosage formof the present application, containing suspended particles. A semisolidis not pourable; it does not flow or conform to its container at roomtemperature. A semisolid does not flow at low shear stress and generallyexhibits plastic flow behavior. A colloidal dispersion is a system inwhich particles of colloidal dimension (i.e., typically between 1 nm and1 μm) are distributed uniformly throughout a liquid.

In some embodiments, “gel” is a semisolid system consisting either ofsuspensions of small inorganic particles or of organic moleculesinterpenetrated by a liquid. “Gels” are classed either as single-phaseor two-phase systems. “Gels” also consist of a mesophase, or state ofmatter intermediate between a liquid and a solid that represents apartially ordered structure, which is the state for the active agents inthe “Gel Drop” of the present embodiments. A two-phase gel consists of anetwork of small discrete particles. In a two-phase system, the gel masssometimes is referred to as magma (e.g., Bentonite Magma) if theparticle size of the suspended material is large. Both gels and magmasare thixotropic, forming semisolids on standing and becoming liquid onagitation. The semisolid formulations should be shaken beforeadministration to ensure homogeneity and should be so labeled (seeSuspensions). Single-phase gels consist of organic macromoleculesuniformly distributed throughout a liquid in such a manner that noapparent boundaries exist between the dispersed macromolecules and theliquid. Single phase gels may also consist of organic low molecularweight (LMW) molecules where the component responsible for gelation isthe actual active ingredient. These so called “LMW hydrogels” aredifferent from traditional gelators of water such as high molecularweight synthetic polymers, polysaccharides, and proteins. High molecularweight gelators are highly ordered and uni-directional due to hydrogenbonding whereas the forces governing LMW hydrogels are largelynon-directional van der Waals forces (hydrophobic) interactions. Inpractice LMW hydrogels are observed as highly anisotropic (typicallyfibrillar) structures that propagate throughout the liquid yielding aphysically branched or entangled network. The gels can thus benon-ordered to slightly ordered showing some birefringence, liquidcrystal character. Gels are administered topically or, after shaking, inthe form of a hydrogel as an eye drop.

The semisolid “gel” according to the present application is a semisolidper USP definitions and literature referenced therein. The semisolidformulation apparent viscosity increases with concentration. Theclinical dosage strength of the present formulation ranges from a lowstrength of ≦1 mg/mL (0.1%) to a high strength of ≦6 mg/mL (0.6%). Lowstrength doses are least viscous and fall under the category of a“solution,” whereas higher strengths are more viscous and fit thedefinition of a gel.

“Jelly” according to the present application is a class of gels, whichare semisolid systems that consist of suspensions made up either smallinorganic particles or large organic molecules interpenetrated by aliquid, in which the structural coherent matrix contains a high portionof liquid, usually water.

“Solution” according to the present application is a clear, homogeneousliquid dosage form that contains one or more chemical substancesdissolved in a solvent or mixture of mutually miscible solvents. Asolution is a liquid preparation that contains one or more dissolvedchemical substances in a suitable solvent or mixture of mutuallymiscible solvents. Because molecules of a drug substance in solution areuniformly dispersed, the use of solutions as dosage forms generallyprovides assurance of uniform dosage upon administration and goodaccuracy when the solution is diluted or otherwise mixed.

“Liquid” according to the present application is a dosage formconsisting of a pure chemical in its liquid state. A liquid is pourable;it flows and conforms to its container at room temperature. Liquidsdisplay Newtonian or pseudoplastic flow behavior.

“Suspension” according to the present application is a liquid dosageform that contains solid particles dispersed in a liquid vehicle.

The compounds of first active agents (e.g., Formula I or II) and thesecond active agents are formulated into therapeutic formulations asnatural or salt forms. Pharmaceutically acceptable non-toxic saltsinclude the base addition salts (formed with free carboxyl or otheranionic groups) which are derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine,2-ethylamino-ethanol, histidine, procaine, and the like. Such salts areformed as acid addition salts with any free cationic groups andgenerally are formed with inorganic acids such as, for example,hydrochloric, sulfuric, or phosphoric acids, or organic acids such asacetic, citric, p-toluenesulfonic, methanesulfonic acid, oxalic,tartaric, mandelic, and the like. Salts of the application include aminesalts formed by the protonation of an amino group with inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid, and the like. Salts of the application alsoinclude amine salts formed by the protonation of an amino group withsuitable organic acids, such as p-toluenesulfonic acid, acetic acid, andthe like. Additional excipients which are contemplated for use in thepractice of the present application are those available to those ofordinary skill in the art, for example, those found in the United StatesPharmacopoeia Vol. XXII and National Formulary Vol. XVII, U.S.Pharmacopoeia Convention, Inc., Rockville, Md. (1989), the relevantcontents of which is incorporated herein by reference. In addition,polymorphs of the application compounds are included in the presentapplication.

The embodiments of the present application provide an ophthalmiccomposition or formulation for treating ocular neovascularization with afirst active agent of Formula I:

or a pharmaceutically acceptable salt thereof; a second active agent ora pharmaceutically acceptable salt thereof, wherein the second activeagent is nicotinic acid, nicotinamide, or vitamin K, or a combinationthereof; and pharmaceutically acceptable excipients; the first activeagent or the pharmaceutically acceptable salt is present in about 0.02%to about 1.2% w/v, wherein:

X¹ is O or S;

R¹ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C(O)(C₁-C₁₀alkyl), (CH₂)_(t)(C₆-C₁₀ aryl), (CH₂)_(t) (4-10 membered heterocyclic),C(O)(CH₂)_(t)(C₆-C₁₀ aryl), or C(O)(CH₂)_(t) (5-10 memberedheterocyclic), wherein:

-   -   t is an integer from 0 to 5;    -   the alkyl group optionally includes 1 or 2 hetero moieties        selected from O, S and N(R⁶) with the proviso that two O atoms,        two S atoms, or an O and an S atoms are not attached directly to        each other;    -   the aryl and heterocyclic groups are optionally fused with a        C₆-C₁₀ aryl group, a C₅-C₈ saturated cyclic group, or a 5-10        membered heterocyclic group;    -   1 or 2 carbon atoms in the foregoing heterocyclic moieties are        optionally substituted with an oxo (═O) moiety or an anion of        oxygen;    -   the (CH₂)_(t) moieties optionally include a carbon-carbon double        or triple bond when t is an integer from 2 to 5; and    -   the foregoing R¹ groups, except H, are optionally substituted        with 1 to 3 R⁴ groups;

R² is H;

R³ is (CH₂)_(t)(C₆-C₁₀ aryl), wherein:

-   -   t is an integer from 0 to 5;    -   the aryl group is optionally fused with a C₆-C₁₀ aryl group, a        C₅-C₈ saturated cyclic group, or a 5-10 membered heterocyclic        group;    -   the (CH₂)_(t) moieties optionally include a carbon-carbon double        or triple bond when t is an integer from 2 to 5; and

each R⁴ is independently selected from C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, halo, cyano, nitro, trifluoromethyl, trifluoromethoxy,azido, OR⁵, C(O)R⁵, C(O)OR⁵, NR⁶C(O)R⁵, NR⁶C(O)OR⁵, OC(O)R⁵, NR⁶SO₂R⁵,SO₂NR⁵R⁶, C(O)NR⁵R⁶, NR⁵R⁶, S(O)_(j)R⁷ where j is an integer from 0 to2, SO₃H, NR⁵(CR⁶R⁷)_(t)OR⁶, (CH₂)_(t)(C₆-C₁₀ aryl), SO₂(CH₂)_(t)(C₆-C₁₀aryl), S(CH₂)_(t)(C₆-C₁₀ aryl), O(CH₂)_(t)(C₆-C₁₀ aryl), (CH₂)_(t) (5-10membered heterocyclic), and (CR⁶R⁷)_(m)OR⁶, wherein:

-   -   m is an integer from 1 to 5;    -   t is an integer from 0 to 5;    -   the alkyl group optionally includes 1 or 2 hetero moieties        selected from O, S and N(R⁶) with the proviso that two O atoms,        two S atoms, or an O and an S atoms are not attached directly to        each other;    -   the aryl and heterocyclic groups are optionally fused with a        C₆-C₁₀ aryl group, a C₅-C₈ saturated cyclic group, or a 5-10        membered heterocyclic group;    -   1 or 2 carbon atoms in the foregoing heterocyclic moieties are        optionally substituted with an oxo (═O) moiety or an anion of        oxygen; and    -   the alkyl, aryl and heterocyclic moieties of the foregoing R⁴        groups are optionally substituted with 1 to 3 substituents        independently selected from halo, cyano, nitro, trifluoromethyl,        trifluoromethoxy, azido, NR⁶SO₂R⁵, SO₂NR⁵R⁶, C(O)R⁵, C(O)OR⁵,        OC(O)R⁵, NR⁶C(O)R⁵, C(O)NR⁵R⁶, NR⁵R⁶, (CR⁶R⁷)_(m)OR⁶ where m is        an integer from 1 to 5, OR⁵, and the substituents listed in the        definition of R⁵; and

R⁵, R⁶, and R⁷ are each independently H or C₁-C₆ alkyl.

In one embodiment, R³ is (CH₂)_(t)(C₆-C₁₀ aryl), wherein t is an integerfrom 1 to 3 and R³ is optionally substituted with 1 to 4 R⁴ groups.

In a further embodiment, R³ is benzyl, optionally substituted with 1 to4 substituents independently selected from halo and C₁-C₄ alkyl. In afurther embodiment, R³ is benzyl substituted with 1 to 4 substituentsindependently selected from methyl, fluoro, chloro and bromo.

In one embodiment, R¹ is (CH₂)_(t) (5-10 membered heterocyclic), whereint is an integer from 0 to 5, optionally substituted with 1 or 2substituents independently selected from C₁-C₄ alkyl, hydroxy andhydroxymethyl.

The present application provides heterocyclic moiety of the R¹ group inFormula I selected from morpholino, pyrrolidinyl, imidazolyl,piperazinyl, piperidinyl, and 2,5-diaza-bicyclo[2.2.1]hept-2-yl, the tvariable of the R¹ group ranges from 2 to 5, and the R¹ group isoptionally substituted with one or more hydroxy groups.

For example, the heterocyclic moiety of the R¹ group in Formula I of thepresent application is pyrrolidine.

In further embodiments of the present application, the first activeagent is:

A compound of the present application is3-[(4-bromo-2,6-difluorophenyl)methoxy]-5-[[[[4-(1-pyrrolidinyl)butyl]amino]carbonyl]amino]-4-isothiazolecarboxamidehydrochloride, of molecular formula: C₂₀H₂₄BrF₂N₅O₃S.HCl, molecularweight: 568.86 g/mol, and with the property that the molecule does notcontain an asymmetric center and is not chiral. A compound of thepresent application is represented by Compound-I:

The Compound-I of the present application is an inhibitor of thetyrosine kinase activity of VEGFR-2, which blocks VEGF-stimulatedauto-phosphorylation of this receptor as well as endothelial cellproliferation. It is selective (>500×) relative to the concentrationrequired to inhibit the epidermal growth factor receptor (EGFR) and theinsulin receptor (IR) tyrosine kinases. Compound-I is described in U.S.Pat. No. 6,235,764. In some embodiments, the compounds of Formula I orII are VEGFR-2 inhibitors.

The second active agents of the present application are EGFR modulators(e.g., activators) of the tyrosine kinase activity of EGFR.

The first active agent and the second active agent can be administeredtogether as part of the same formulation comprising the first activeagent, the second active agent, and a pharmaceutical excipient. Thefirst active agent and the second active agent can also be administeredseparately. In one embodiment, the first active agent and the secondactive agent are administered separately as two formulations, whereinone formulation comprises the first active agent and a pharmaceuticalexcipient, and the second formulation comprises the second active agentand a pharmaceutical excipient.

In one embodiment, a compound of Formula I or II, or a pharmaceuticallyacceptable salt thereof, and the second active agent or apharmaceutically acceptable salt thereof, are administeredsimultaneously. Alternatively, a compound of Formula I or II, or apharmaceutically acceptable salt thereof, is administered prior toadministration of the second active agent, or a pharmaceuticallyacceptable salt thereof. In another embodiment, a compound of Formula Ior II, or a pharmaceutically acceptable salt thereof, is administeredafter administration of the second active agent, or a pharmaceuticallyacceptable salt thereof.

General Properties

Compound-I of the present application has the characteristics as shownin Table 1. The embodiments provide three formulations of Compound-I orits free base—the Formula II compound.

TABLE 1A General Properties of Compound-I Drug Substance Property ResultChemical Name [CAS No] 3-[(4-bromo-2,6- Codes: Compound-Idifluorophenyl)methoxy]-5-[[[[4- (1-pyrrolidinyl)butyl]amino]carbonyl]amino]-4- isothiazolecarboxamide hydrochloride [252003-71-7]Appearance (color, physical form) White crystalline solid Melting range222.2-224.8° C. pKa (water) 10.5 Solubility (mg/mL) Methanol: 4.3Ethanol: 0.7 Acetonitrile: 0.04 Tetrahydrofuran: 0.02 Hexanes: <0.010.1N NaOH: 0.05 pH 9.0 (0.05m Na₂HPO₄): 0.7 pH 7.5 (0.05M NaH₂PO₄): 1.00.1N HCl: 0.04 Deionized water 0.5-1.2^(b)

The composition of Compound-I formulations are listed in Table 1B. Theformulation materials are listed in Table 1C.

TABLE 1B Compound-I formulations: Gel Drop, suspension, and solution.Formulation Forms Composition Ophthalmic gel drops 0.05% SodiumPhosphate, Monobasic, Monohydrate 1.0-2.0% Glycerin with or without0.005% Benzalkonium Chloride, NF (BAK) pH ~6.0-7.0 Tris-basedsuspensions 0.6% Tromethamine, USP (Tris) 1.0-2.0% Glycerin, USP with orwithout 0.005% Benzalkonium Chloride, NF (BAK) pH ~6.0-7.0Cyclodextrin-based 1% to 20% hydroxypropyl-β-cyclodextrin solutions(HP-β-CD, KLEPTOSE ® HPB) 0.1% to 0.9% sodium chloride pH ~6.0-7.0 or 1%to 20% sulfobutylether-β-cyclodextrin (SBE-β-CD, CAPTISOL ®) with orwithout 0.122% Tromethamine (Tris) 0.1-0.2% sodium phosphate, dibasic,anhydrous 0%-0.6% sodium chloride pH ~6.0-7.0 or 1% to 20% HP-β-CD(KLEPTOSE ® HPB or KLEPTOSE ® HP) 0.1-0.2% sodium phosphate, dibasic,anhydrous 0.50%-0.6% sodium chloride pH ~6.0-7.0

TABLE 1C Formulation materials Material Function Compound-I Active DrugSubstance Sodium Chloride Tonicity Modifier Sulfobutylether-β-cyclodextrin Solubilizing (CAPTISOL ®, SβECD) agents2-hydroxypropyl-β-cyclodextrin (KLEPTOSE ® HPB Parenteral Grade, HPβCD)Trometamol (Tris) Buffer Dibasic phosphate buffer 2.0N NaOH Adjust pH0.1N HCl

The composition of the Compound-I formulations and a second active arelisted in Table 1D. The formulation materials are listed in Table 1E.

TABLE 1D Compound-I formulations: Gel Drop, suspension, and solution.Formulation Forms Composition Ophthalmic gel drops 0.05% SodiumPhosphate, Monobasic, Monohydrate 1.0-2.0% Glycerin with or without0.005% Benzalkonium Chloride, NF (BAK) pH ~6.0-7.0 Tris-basedsuspensions 0.6% Tromethamine, USP (Tris) 1.0-2.0% Glycerin, USP with orwithout 0.005% Benzalkonium Chloride, NF (BAK) pH ~6.0-7.0Cyclodextrin-based 1% to 20% hydroxypropyl-β-cyclodextrin solutions(HP-β-CD, KLEPTOSE ® HPB) 0.1% to 0.9% sodium chloride pH ~6.0-7.0 or 1%to 20% sulfobutylether-β-cyclodextrin (SBE-β-CD, CAPTISOL ®) with orwithout 0.122% Tromethamine (Tris) 0.1-0.2% sodium phosphate, dibasic,anhydrous 0%-0.6% sodium chloride pH ~6.0-7.0 or 1% to 20% HP-β-CD(KLEPTOSE ® HPB or KLEPTOSE ® HP) 0.1-0.2% sodium phosphate, dibasic,anhydrous 0.50%-0.6% sodium chloride pH ~6.0-7.0

TABLE 1E Formulation materials Material Function Compound-I Active DrugSubstance Second active agent Active Drug substance Sodium ChlorideTonicity Modifier Sulfobutyl ether-β-cyclodextrin Solubilizing agents(CAPTISOL ®, SβECD) 2-hydroxypropyl-β-cyclodextrin (KLEPTOSE ® HPBParenteral Grade, HPβCD) Trometamol (Tris) Buffer Dibasic phosphatebuffer 2.0N NaOH Adjust pH 0.1N HClOphthalmic Solutions

The present application provides formulations of a first active agent(e.g., Compound-I and/or its free base (Formula II compound)) and/or asecond active agent, formed as a solution with viscosity similar towater. The solution includes pharmaceutically acceptableagents/excipients, for example, without being limiting, cyclodextrin.The solution thus formed is clear and colorless solution, suitable fortopical administration to the eye.

The solutions of the present application reduce anterior segmentexposure of the first active agent; thereby they allow increasedconcentration of the first active agent, e.g., a compound of Formula Ior II, in the solution and increased frequency of delivery, thus,promoting maintained high concentration of the first active agent in theposterior segment of the eye.

The solutions of the application comprise about 0.005% to about 5.0% w/vof the first active agent of Formula I or II, or a pharmaceuticallyacceptable salt thereof, for example, Compound-I. In some embodiments,the concentration of Compound-I or its free base (Formula II) in thesolutions is about 0.005%-about 0.01%, about 0.01%-about 0.05%, about0.05%-about 0.1%, about 0.1%-about 0.2%, about 0.2%-about 0.3%, about0.3%-about 0.4%, about 0.4%-about 0.5%, about 0.5%-about 0.6%, about0.6%-about 0.7%, about 0.7%-about 0.8%, about 0.8%-about 0.9%, about0.9%-about 1.0%, about 1.0-about 2.0%, about 2.0-about 3.0%, about3.0-about 4.0%, or about 4.0-about 5.0% w/v for topical administration.In some embodiments, the concentration of Compound-I or its free base(Formula II) in the formulations is about 0.1%-about 1.2%, about0.2%-about 1.2%, about 0.3%-about 1.2%, about 0.4%-about 1.2%,0.1%-about 1.1%, about 0.2%-about 1.1%, about 0.3%-about 1.1%, about0.4%-about 1.1%, 0.1%-about 1.0%, about 0.2%-about 1.0%, about0.3%-about 1.0%, about 0.4%-about 1.0%, 0.1%-about 0.8%, about0.2%-about 0.8%, about 0.3%-about 0.8%, about 0.4%-about 0.8%,0.1%-about 0.6%, about 0.2%-about 0.6%, about 0.3%-about 0.6%, about0.4%-about 0.6%, 0.1%-about 0.5%, about 0.2%-about 0.5%, about0.3%-about 0.5%, about 0.4%-about 0.5%, 0.1%-about 0.4%, about0.2%-about 0.4%, about 0.3%-about 0.4% w/v for topical administration.In some embodiments, the solutions include about 0.005%, about 0.05%,about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%,about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 2.0%, about 3.0%,about 4.0%, or about 5.0% w/v of Compound-I or its free base (FormulaII).

The solutions of the application may further comprise about0.00001%-about 5.0% w/v of a second active agent, or a pharmaceuticallyacceptable salt thereof, for example, nicotinic acid, nicotinamide, orvitamin K, or a combination thereof. The solutions of the applicationmay further comprise is about 0.00001%-about 1.0%, about 0.00001%-about0.1%, about 0.00001%-about 0.01%, about 0.00001%-about 0.001%, about0.00001%-about 0.0002%, or about 0.00001%-about 0.0001% w/v of a secondactive agent, or a pharmaceutically acceptable salt thereof, forexample, nicotinic acid, nicotinamide, or vitamin K, or a combinationthereof. In some embodiments, the concentration of the second activeagent in the solutions is about 0.00001%-about 0.0001%, 0.000012%-about0.0001%, 0.000014%-about 0.0001%, 0.000016%-about 0.0001%,0.000018%-about 0.0001%, 0.00002%-about 0.0001%, 0.00003%-about 0.0001%,0.00004%-about 0.0001%, 0.00005%-about 0.0001%, 0.00006%-about 0.0001%,0.00007%-about 0.0001%, 0.00008%-about 0.0001%, 0.00009%-about 0.0001%,0.000016%-about 0.00009%, 0.000018%-about 0.00009%, 0.00002%-about0.00009%, 0.00003%-about 0.00009%, 0.00004%-about 0.00009%,0.00005%-about 0.00009%, 0.00006%-about 0.00009%, 0.00007%-about0.00009%, 0.00008%-about 0.00009% w/v for topical administration. Insome embodiments, the solutions include about 0.00001%, 0.00002%,0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.000081%,0.000082%, 0.000083%, 0.000084%, 0.000085%, 0.000086%, 0.000087%,0.000088%, or 0.000089% w/v of the second active agent.

The solutions of the application may further comprise about 0.5 μM,about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM,about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM,about 8 μM, or about 9 μM of a second active agent, or apharmaceutically acceptable salt thereof, for example, nicotinic acid,nicotinamide, or vitamin K, or a combination thereof. In someembodiments, the concentration of the second active agent is about 1 μM.

In some embodiments, the formulation comprises cyclodextrin forimproving solubility of a first active agent (e.g., Compound-I).Cyclodextrin, an oligosaccharide made up of six to eight dextrose unitsjoined through one or four bonds increases solubility of active agentsthat have poor or low solubility in water or aqueous solutions (e.g., inPBS buffer). Cyclodextrins form hydrophilic complexes with hydrophobicactive agents.

One or more cyclodextrins may be used in the solution of the presentapplication. Non-limiting examples of cyclodextrins for use informulation of the present application are, for example:2-hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, randomlymethylated-β-cyclodextrin, ethylated-β-cyclodextrin,triacetyl-β-cyclodextrin, peracetylated-β-cyclodextrin,carboxymethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin,2-hydroxy-3-(trimethylammonio)propyl-β-cyclodextrin,glucosyl-β-cyclodextrin, maltosyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, branched-β-cyclodextrin,hydroxypropyl-γ-cyclodextrin, randomly methylated-γ-cyclodextrin,trimethyl-γ-cyclodextrin, or a combination thereof.

In some embodiments, the solution of Formula II compound or Compound-Icomprising cyclodextrin is a clear and colorless solution and has aviscosity similar to water. In some embodiments, the present applicationprovides a solution comprising Compound-I, one or more cyclodextrin, anda second active agent for topical application and is topically appliedto the eye.

The ophthalmic solution of the present application comprisescyclodextrin and pharmaceutical excipients chosen at or belowconcentrations optimal for ophthalmic solution. The excipients of thepresent application are, for example, benzalkonium chloride (BAK) andNaCl. In some embodiments, the ophthalmic solution comprises about0.001-about 0.005% w/v Benzalkonium chloride (BAK). The BAK amountvaries depending on the need of the application.

The ophthalmic solution comprises, for example, without being limiting,about 0.005%-5.0% Compound-I or its free base, about 2-about 25%cyclodextrin, e.g., without being limiting, Hydroxypropyl-β-cyclodextrin(HPβCD) or methylcyclodextrin (KLEPTOSE® HPB), and/or sulfobutylether-β-cyclodextrin (CAPTISOL®), about 0.1-about 0.7% salt, e.g.,without being limiting, NaCl, and/or about 0.005% of an anti-microbialagent, for example, without being limiting, Benzalkonium chloride (BAK).The formulation comprises Compound-I or its free base to cyclodextrinratio 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or between 1:10 and1:20. In some embodiments, the ophthalmic solution comprisingcyclodextrin further comprises tromethamine (also known as Tris,Tris(Hydroxymethyl)aminomethane, or Tris buffer). In some embodiments,the ophthalmic solution comprises about 0.05%-1% Tris. In someembodiments, the ophthalmic solution comprises about 0.05%-0.5% Tris. Insome embodiments, the ophthalmic solution comprises about 0.05%-0.2%Tris. In some embodiments, the ophthalmic solution comprises about0.1%-0.15% Tris. In some embodiments, the ophthalmic solution comprisesabout 1% Tris. In some embodiments, the ophthalmic solution furthercomprises about 0.005%-5.0% second active agent.

Ophthalmic solutions of the present embodiments include, for example,without being limiting: about 0.3%-about 5.0% Compound-I (about 3mg/mL-about 50.0 mg/mL), about 0.05% sodium phosphate monobasicmonohydrate, about 2% glycerin; about 0.4% Compound-I, about 7% HPβCD,about 0.7% NaCl, about 0.005% BAK; about 0.4% Compound-I, about 4%HPβCD, about 0.7% NaCl, about 0.005% BAK; about 0.4% Compound-I, about7% HPβCD, about 1% tromethamine, about 0.4% NaCl, about 0.005% BAK; andabout 0.6% Compound-I, about 7% HPβCD, about 0.7% NaCl, about 0.005%BAK. For Compound-I of between about 0.005% to about 5.0%concentrations, cyclodextrin is present at a corresponding molar ratio.In some embodiments, the ophthalmic solution further comprises about0.005%-5.0% second active agent.

Additional ophthalmic solutions include, for example, without beinglimiting: about 0.4% Formula II compound (free base), about 7.15% HPβCD,about 0.7% NaCl; about 0.1% Formula II compound (free base), about 1.79%HPβCD, about 0.85% NaCl; about 0.2% Formula II compound (free base),about 3.57% HPβCD, about 0.8% NaCl; about 0.6% Formula II compound (freebase), about 10.72% HPβCD, about 0.6% NaCl; about 0.4% Formula IIcompound (free base), about 8.41% HPβCD, about 0.65% NaCl; about 0.4%Compound-I, about 10.51 HPβCD, about 0.65% NaCl; about 0.4% Formula IIcompound (free base), about 10.51% HPβCD, about 0.15% NaCl, about 1.0%tromethamine (Tris); and/or about 0.1% Formula II compound (free base),about 20.63% HPβCD, about 0.8% NaCl; about 0.6% Compound-I (as freebase), about 15.77% HPβCD, about 0.37% NaCl. For Formula II of betweenabout 0.005% to about 5.0% concentrations, cyclodextrin is present at acorresponding molar ratio. In some embodiments, the ophthalmic solutionfurther comprises about 0.005%-5.0% second active agent.

In some embodiments, the ophthalmic solutions include between about1.0%-about 25% cyclodextrin. For example, without being limiting, theCompound-I formulations include about 2.0%-about 3.0% HPβCD, about3.0%-about 5.0% HPβCD, about 5.0%-about 10% HPβCD, or about 10%-about25% HPβCD. In some embodiments, the ophthalmic solution furthercomprises about 0.005%-5.0% second active agent.

In additional embodiments, the ophthalmic solutions are formulated as,for example, without being limiting: about 8.41% KLEPTOSE® HPB and about0.142% phosphate; about 8.9% KLEPTOSE® HPB and about 0.142% phosphate;about 4.88% CAPTISOL® and about 0.142 phosphate; and/or about 4.88%CAPTISOL® and about 0.122% phosphate.

In some embodiments, the ophthalmic solutions comprising cyclodextrinsare clear and colorless, and are extremely viscous, moderately viscous,or have viscosity similar to water.

In some embodiments, the ophthalmic solution of the application has a pHvalue of about 4.5 to about 7.5 at or under about 40° C.

In some embodiments, the ophthalmic solution of the application has a pHvalue of about 5.0 to about 7.0 at or under about 40° C.

For example, the ophthalmic solution of the application has a pH valueof about 6.0 at or under about 40° C.

The ophthalmic solutions of the present application may contain variousadditives incorporated ordinarily, such as buffering agents (e.g.,phosphate buffers, borate buffers, citrate buffers, tartrate buffers,acetate buffers, amino acids, sodium acetate, sodium citrate and thelike), tonicity agents (e.g., saccharides such as sorbitol, glucose andmannitol, polyhydric alcohols such as glycerin, concentrated glycerin,PEG and Propylene glycol, salts such as sodium chloride, etc.),preservatives or antiseptics (e.g., Benzalkonium chloride, Benzatkoniumchloride, P-oxybenzoates such as Methyl p-oxybenzoate or Ethylp-oxybenzoate, Benzyl alcohol, Phenethyl alcohol, Sorbic acid or itssalt, Thimerosal, Chlorobutanol, etc.), solubilizing aids or particlestabilizing agents (e.g., water-soluble polymers such as polyvinylpyrrolidone, surfactants such as tyloxapol, polysorbates, poloxamer,etc.), pH modifiers (e.g., Hydrochloric acid, Acetic acid, Phosphoricacid, Sodium hydroxide, Potassium hydroxide, Ammonium hydroxide and thelike), thickening agents (e.g., HEC, Hydroxypropyl cellulose, Methylcellulose, HPMC, Carboxymethyl cellulose and their salts), chelatingagents (e.g., sodium edetate, sodium citrate, condensed sodium phosphateetc.), and second active agent stabilizers (e.g., EDTA, propyl gallate,and a combination thereof).

The ophthalmic solutions of the present application comprisecyclodextrin, and may further comprise additional excipients, forexample, without being limiting, about 0.5%-about 3% surfactant andemulsifier, for example, without being limiting, polysorbate 80 orequivalent excipients thereof; about 0.05-about 0.4% nonionic liquidpolymer of the alkyl aryl polyether alcohol type, for example, withoutbeing limiting tyloxapol; and/or about 0.05%-about 0.6% hydrophilicnon-ionic surfactant, for example, without being limiting, poloxamer,such as poloxamer 407.

In some embodiments, the ophthalmic solution comprises about 0.01-about0.5%, about 0.02-about 0.5%, about 0.04-about 0.5%, about 0.06-about0.5%, about 0.08-about 0.5%, about 0.08-about 0.4%, about 0.08-about0.3%, about 0.08-about 0.2%, about 0.08-about 0.18%, about 0.08-about0.16%, about 0.08-about 0.14%, or about 0.08-about 0.12% EDTA. In someembodiments, the ophthalmic solution comprises about 0.04%, about 0.06%,about 0.08%, about 0.1%, about 0.12, about 0.14%, about 0.16%, about0.18%, or about 0.2% EDTA. In some embodiments, the ophthalmic solutioncomprises about 0.1% EDTA.

In some embodiments, the ophthalmic solution comprises about 0.001-about0.5%, about 0.002-about 0.5%, about 0.005-about 0.5%, about 0.01-about0.5%, about 0.02-about 0.5%, about 0.03-about 0.5%, about 0.04-about0.5%, about 0.01-about 0.4%, about 0.01-about 0.3%, about 0.01-about0.2%, about 0.01-about 0.1%, about 0.01-about 0.08%, about 0.01-about0.06% propyl gallate. In some embodiments, the ophthalmic solutioncomprises about 0.01%, about 0.02%, about 0.03%, about 0.04%, about0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09% propylgallate. In some embodiments, the ophthalmic solution comprises about0.05% propyl gallate.

Concentration in Various Ocular Tissues—Delivered as an OphthalmicSolution

The ocular solution comprising cyclodextrin improves bioavailability ofthe first active agents of the present application at the posteriorsegment of the eye. Without being bound by theory, in an embodiment, theformulation comprising cyclodextrin forms a clear and colorlesssolution, which lowers corneal exposure of the active agent, forexample, exposure of Compound-I, by about 5-15 fold compared to thecorneal exposure to with an equimolar Gel Drop formulation.

Without being bound by theory, in one embodiment, an ophthalmic solutioncomprising cyclodextrin increases the therapeutic index of Compound-Iduring topical ocular administration. Upon administration, thehydrophilic complex of cyclodextrin-Compound-I is pharmacologicallyinert at the cornea. Without being bound by theory, in some embodiments,the cyclodextrin-Compound-I complex increases corneal tolerability ofCompound-I. Without being bound by theory, in some embodiments,spontaneous dissociation of cyclodextrin from Compound-I at theperipheral vasculature increases bioavailability at the target tissue,e.g., at the choroid or retina.

Unlike other formulations of Compound-I, which in some embodimentscontribute to corneal toxicity, the cyclodextrin-based ophthalmicsolution comprising similar concentration of Compound-I lower cornealexposures and, thereby increase the therapeutic index and correspondingbenefits to patients. In one embodiment, the use of cyclodextrin-basedsolution of Compound-I provides approximately 10× reduction in cornealexposure, as compared to equimolar concentrations of Gel Drop. In someembodiments, the cyclodextrin-based solution of Compound-I reducescorneal exposure of Compound-I by 5×, 20×, 30×, 40×, or 50×. In oneembodiment, 1-90 days or 3-9 months of topical ocular dosing of about0.005%-about 5.0% Compound-I as a cyclodextrin-based solution does nothave any adverse or toxic effect at the cornea, choroid, and/or theretina. In yet another embodiment, 1-90 days of topical ocular dosing ofabout 0.6%-about 5.0% Compound-I as a cyclodextrin-based solution doesnot have any adverse or toxic effect at the cornea, choroid, and/or theretina.

The lowering of the corneal exposure is correlated with increasingbioavailability and therapeutic index of the active agent at theposterior segment, for example, at the retina or choroid, of the eye.For example, no toxic effect attributable to the first active agent or asuitable carrier is observed to the cornea or other parts of the eyewhen about 0.1%-about 5.0% Compound-I formulation comprisingcyclodextrin is administered topically administered to the eye for atleast 30 days or more than 60 days.

In one embodiment, when a formulation comprising about 0.4% (about 4mg/mL) of Compound-I or its free base, and cyclodextrin, whenadministered topically to the eye, the central choroid concentration isbetween about 0.2 μM-about 0.9 μM, central retina concentration of theactive agent is between about 0.02 μM-about 0.4 μM, aqueous humorconcentration of the active agent is about 0.003 μM-about 0.009 μM, andcorneal concentration of the active agent is between 6 μM-40 μM. Thecyclodextrin used in the formulation is, for example, without beinglimiting example, KLEPTOSE® HPB or CAPTISOL®.

In some embodiments, a cyclodextrin-based solution of Compound-I or itsfree base increases the bioavailability of the active agent at thecentral choroid and the central retina, while reducing concentration atthe cornea. In some embodiments, topical delivery of Compound-I or itsfree base formulated in the presence of cyclodextrin reduces the cornealconcentration by about 5-about 15 fold over the corneal concentration ofequimolar Gel Drop.

Without being bound by theory, in some embodiments, the combined effectsof decreasing corneal drug exposure so as to avoid poor oculartolerability while increasing posterior segment bioavailability increasethe therapeutic index and corresponding benefits to patients.

In some embodiments, the exposure time of Compound-I and the secondactive agent is between 1 and 90 days or for longer than 90 days (e.g.,4 months, 6 months, 8 months, or 12 months). In some embodiments, thedosage regimen involves several courses of topical ocular administrationof a formulation comprising Compound-I and a second active agent,wherein the second active agent is administered as a separateformulation or as part of the Compound-I formulation, to a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). For example, the dosage regimeninvolves once daily, twice daily, three times daily or four times dailyadministration of the formulation for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).For example, the dosage regimen involves once, twice, three times, orfour times administration of a compound of Formula I or II and a secondactive agent on alternate days (i.e., on day 1, 3, 5, 7 etc.) for up to90 days. For example, the dosage regimen involves administering once onday 1, once or twice on day 2-day 90. For example, the dosage regimeninvolves administering once, twice, three times, or four times on day 1,followed by once daily for 2-90 days. For example, the dosage regimeninvolves administering once, twice, three times, four times on day 1,followed by once, twice, three times, or four times on alternate days(i.e., on day 1, 3, 5, 7 etc.) for up to 90 days. For example, onedosage regimen involves once per day or twice per day for 1, 2, 3, 4, or5 consecutive days. For twice or three daily dosage regimen, subjectsreceive topical ocular dose of a Compound-I formulation and a secondactive agent on days 1 and 4 approximately about 4, 6, or 8 hours apart.In another embodiment, subjects receive topical ocular doses of aCompound-I formulation and a second active agent approximately about 4,6, or 8 hours apart for four consecutive days. In some embodiments,subjects receive one or two doses of topical ocular dose of Compound-Iformulation and a second active agent per day for 5 consecutive days. Inyet other embodiments, subjects receive one or two doses of topicalocular dose of Compound-I and a second active agent formulation for 5-90consecutive days. In some embodiments, subjects receive one or two dosesof topical ocular dose of Compound-I formulation and a second activeagent for at least 25 consecutive days. In one embodiment, subjectsreceive one or two topical ocular doses for at least 90 consecutive daysor more. The second active agent can be administered separately or aspart of the Compound-I formulation. When administered separately, thesecond active agent, or a pharmaceutical salt thereof, can beadministered alone or as a formulation.

For example, a formulation comprising about 1 mg/mL BID of a firstactive agent (e.g., Compound-I) and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments a formulation comprising about 1 mg/mLQD of a first active agent (e.g., Compound-I) and a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 1mg/mL TID of a first active agent (e.g., Compound-I) and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments a formulation comprisingabout 1 mg/mL QID of a first active agent (e.g., Compound-I) and asecond active agent is administered to one eye or both eyes of a subjectfor between 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 2 mg/mL BID of a first active agent (e.g., Compound-I)and a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 2 mg/mL QD of a first active agent (e.g.,Compound-I) and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodimentsa formulation comprising about 2 mg/mL TID of a first active agent(e.g., Compound-I) and a second active agent is administered to one eyeor both eyes of a subject for between 1 and 90 days or for longer than90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 2 mg/mL QID of a first activeagent (e.g., Compound-I) and a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 3 mg/mL BID of afirst active agent (e.g., Compound-I) and/or a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments a formulation comprising about 3 mg/mLQD of a first active agent (e.g., Compound-I) and/or a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments a formulation comprisingabout 3 mg/mL TID of a first active agent (e.g., Compound-I) and asecond active agent is administered to one eye or both eyes of a subjectfor between 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 3 mg/mL QID of a first active agent (e.g., Compound-I)and a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 4 mg/mL BID of a first active agent (e.g.,Compound-I) and/or a second active agent is administered to one eye orboth eyes of a subject for between 1 and 90 days or for longer than 90days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 4 mg/mL QD of a first activeagent (e.g., Compound-I) and/or a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 4 mg/mL TID of afirst active agent (e.g., Compound-I) and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments a formulation comprising about 4 mg/mLQID of a first active agent (e.g., Compound-I) and a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 5mg/mL BID of a first active agent (e.g., Compound-I) and/or a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 5 mg/mL QD of a first active agent (e.g., Compound-I)and/or a second active agent is administered to one eye or both eyes ofa subject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 5 mg/mL TID of a first active agent (e.g.,Compound-I) and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodimentsa formulation comprising about 5 mg/mL QID of a first active agent(e.g., Compound-I) and a second active agent is administered to one eyeor both eyes of a subject for between 1 and 90 days or for longer than90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 6 mg/mL BID of a first activeagent (e.g., Compound-I) and/or a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 6 mg/mL QD of a firstactive agent (e.g., Compound-I) and/or a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments a formulation comprising about 6 mg/mLTID of a first active agent (e.g., Compound-I) and a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 6mg/mL QID of a first active agent (e.g., Compound-I) and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments a formulation comprisingabout 7 mg/mL BID of a first active agent (e.g., Compound-I) and asecond active agent is administered to one eye or both eyes of a subjectfor between 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 7 mg/mL QD of a first active agent (e.g., Compound-I)and a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 7 mg/mL TID of a first active agent (e.g.,Compound-I) and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodimentsa formulation comprising about 7 mg/mL QID of a first active agent(e.g., Compound-I) and a second active agent is administered to one eyeor both eyes of a subject for between 1 and 90 days or for longer than90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 8 mg/mL BID of a first activeagent (e.g., Compound-I) and a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 8 mg/mL QD of a firstactive agent (e.g., Compound-I) and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments a formulation comprising about 8 mg/mLTID of a first active agent (e.g., Compound-I) and a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 8mg/mL QID of a first active agent (e.g., Compound-I) and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments a formulation comprisingabout 9 mg/mL BID of a first active agent (e.g., Compound-I) and asecond active agent is administered to one eye or both eyes of a subjectfor between 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 9 mg/mL QD of a first active agent (e.g., Compound-I)and a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 9 mg/mL TID of a first active agent (e.g.,Compound-I) and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodimentsa formulation comprising about 9 mg/mL QID of a first active agent(e.g., Compound-I) and a second active agent is administered to one eyeor both eyes of a subject for between 1 and 90 days or for longer than90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 10 mg/mL QD of a first activeagent (e.g., Compound-I) and a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).The dosage regimen for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months) may be any of theregimens involving consecutive or alternate days described in theparagraph above. In some embodiments, the formulation of the presentapplication is administered QD, BID, TID, or QID when administered atlow doses (e.g., 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, or 5 mg/mL), and QDor BID at high doses (e.g., 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, or 10mg/mL).

In some embodiments, a 1 mg/mL BID of a first active agent (e.g.,Compound-I) formulation and a second active agent is administered to oneeye or both eyes of a subject for between 1 and 90 days or for longerthan 90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments, a 1 mg/mL QD of a first active agent (e.g., Compound-I)formulation and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodiments,a 2 mg/mL BID of a first active agent (e.g., Compound-I) formulation anda second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments, a 2mg/mL QD of a first active agent (e.g., Compound-I) formulation and asecond active agent is administered to one eye or both eyes of a subjectfor between 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments, a 3 mg/mL BID of afirst active agent (e.g., Compound-I) formulation and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments, a 3 mg/mL QD of a firstactive agent (e.g., Compound-I) formulation and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments, a 4 mg/mL BID of a first active agent(e.g., Compound-I) formulation and a second active agent is administeredto one eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments, a 4 mg/mL QD of a first active agent (e.g.,Compound-I) formulation and a second active agent is administered to oneeye or both eyes of a subject for between 1 and 90 days or for longerthan 90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments, a 5 mg/mL BID of a first active agent (e.g., Compound-I)formulation and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodiments,a 5 mg/mL QD of a first active agent (e.g., Compound-I) formulation anda second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments, a 6mg/mL BID of a first active agent (e.g., Compound-I) formulation and asecond active agent is administered to one eye or both eyes of a subjectfor between 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments, a 6 mg/mL QD of afirst active agent (e.g., Compound-I) formulation and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments, a 7 mg/mL BID of a firstactive agent (e.g., Compound-I) formulation and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments, a 7 mg/mL QD of a first active agent(e.g., Compound-I) formulation and a second active agent is administeredto one eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments, a 8 mg/mL BID of a first active agent (e.g.,Compound-I) formulation and a second active agent is administered to oneeye or both eyes of a subject for between 1 and 90 days or for longerthan 90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments, a 8 mg/mL QD of a first active agent (e.g., Compound-I)formulation and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodiments,a 9 mg/mL BID of a first active agent (e.g., Compound-I) formulation anda second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments, a 9mg/mL QD of a first active agent (e.g., Compound-I) formulation and asecond active agent is administered to one eye or both eyes of a subjectfor between 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments, a 10 mg/mL BID ofa first active agent (e.g., Compound-I) formulation and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments, a 10 mg/mL QD of a firstactive agent (e.g., Compound-I) formulation and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). The dosage regimen for between 1 and 90 days or for longerthan 90 days (e.g., 4 months, 6 months, 8 months, or 12 months) may beany of the regimens involving consecutive or alternate days described inthe paragraph above.

The present application provides cyclodextrin-based solutions containinghydroxypropyl-beta-cyclodextrin (HP-β-CD, KLEPTOSE® HPB) or CAPTISOL®that are well tolerated when administered topically for 30-90 days orfor 4-6 months. In some embodiments, once or twice daily administrationof at about 0.005%-about 5.0% w/v Compound-I or its free base and asecond active agent in a solution containing about 1.0%-about 25%HP-β-CD or CAPTISOL® is well tolerated by the subject.

In some embodiments, the formulation of Formula II or Compound-I and asecond active agent is administered to one eye or both eyes of asubject. For example, about 0.2%-about 1.0% (w/v) of the compound ofFormula II or about 0.1%-1.2% (w/v) of Compound-I formulation and asecond active agent comprising formulation of the present application isadministered once a day (QD), twice a day (BID), three times a day(BID), or four times a day (QID) to one eye or both eyes of a subjectfor between 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments, the Formula II orCompound-I formulation and a second active agent are administered to oneeye or both eyes of a subject. For example, about 0.2%-about 1.0% (w/v)of the compound of Formula II or about 0.1%-1.2% (w/v) of Compound-Iformulation and a second active agent is administered once a day (QD),twice a day (BID), three times a day (BID), or four times a day (QID) toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).

In some embodiments, Formula II compound or Compound-I is complexed witha complexing agent, e.g., cyclodextrin (e.g., KLEPTOSE® HPB (%)) inratio of about 1:8, in which about 2%-13% (w/v) cyclodextrin (e.g.,KLEPTOSE® HPB (%)) is added to the formulation. The formulation mayfurther comprise about 0.1%-about 0.2% buffer, e.g., 10 mM phosphatebuffer. The desired osmolality of the formulation is about 200-about 300mOsm, achieved by adding quantity sufficient to achieve the osmolalitywith a salt, e.g., sodium chloride. The pH of the formulation is about6.0 at or under about 40° C. The dosage regimen for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months) may be any of the regimens involving consecutive or alternatedays described in the paragraph above.

Ophthalmic Suspensions

The present application provides suspensions of a first active agent(e.g., Compound-I) comprising the agent and pharmaceutically acceptableexcipients. The present application also provides suspensions of a firstactive agent (e.g., Compound-I) and a second active agent comprising thefirst active agent, the second active agent and pharmaceuticallyacceptable excipients. For example, Compound-I suspensions and secondactive agent suspensions may include, without being limiting, bufferingagents, acids & bases, for example, without being limiting, HCl andNaOH. In one embodiment, suspensions of Compound-I or its free base mayinclude a buffering agent, for example, without being limiting,tromethamine (Tris). In another embodiment, suspensions of Compound-I orits free base and a second active agent may include a buffering agent,for example, without being limiting, tromethamine (Tris). Thetromethamine-based suspension of Formula II compound or Compound-I and asecond active agent is useful for topical administration to the eye.

The suspensions of the application comprise about 0.005% to about 5.0%w/v of a first active agent of Formula I or II, or a pharmaceuticallyacceptable salt thereof, for example, Compound-I. In some embodiments,the concentration of Compound-I or its free base (Formula II) in thesuspensions is about 0.005%-about 0.01%, about 0.01%-about 0.05%, about0.05%-about 0.1%, about 0.1%-about 0.2%, about 0.2%-about 0.3%, about0.3%-about 0.4%, about 0.4%-about 0.5%, about 0.5%-about 0.6%, about0.6%-about 0.7%, about 0.7%-about 0.8%, about 0.8%-about 0.9%, about0.9%-about 1.0%, about 1.0-about 2.0%, about 2.0-about 3.0%, about3.0-about 4.0%, or about 4.0-about 5.0% w/v for topical administration.In some embodiments, the concentration of Compound-I or its free base(Formula II) in the formulations is about 0.1%-about 1.2%, about0.2%-about 1.2%, about 0.3%-about 1.2%, about 0.4%-about 1.2%,0.1%-about 1.1%, about 0.2%-about 1.1%, about 0.3%-about 1.1%, about0.4%-about 1.1%, 0.1%-about 1.0%, about 0.2%-about 1.0%, about0.3%-about 1.0%, about 0.4%-about 1.0%, 0.1%-about 0.8%, about0.2%-about 0.8%, about 0.3%-about 0.8%, about 0.4%-about 0.8%,0.1%-about 0.6%, about 0.2%-about 0.6%, about 0.3%-about 0.6%, about0.4%-about 0.6%, 0.1%-about 0.5%, about 0.2%-about 0.5%, about0.3%-about 0.5%, about 0.4%-about 0.5%, 0.1%-about 0.4%, about0.2%-about 0.4%, about 0.3%-about 0.4% w/v for topical administration.In some embodiments, the suspensions include about 0.005%, about 0.05%,about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%,about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 2.0%, about 3.0%,about 4.0%, or about 5.0% w/v of Compound-I or its free base (FormulaII).

The suspensions of the application may further comprise about0.00001%-about 5.0% w/v of a second active agent, or a pharmaceuticallyacceptable salt thereof, for example, nicotinic acid, nicotinamide, orvitamin K, or a combination thereof. The suspensions of the applicationmay further comprise about 0.00001%-about 1.0%, about 0.00001%-about0.1%, about 0.00001%-about 0.01%, about 0.00001%-about 0.001%, about0.00001%-about 0.0002%, or about 0.00001%-about 0.0001% w/v of a secondactive agent, or a pharmaceutically acceptable salt thereof, forexample, nicotinic acid, nicotinamide, or vitamin K, or a combinationthereof. In some embodiments, the concentration of the second activeagent in the suspensions is about 0.00001%-about 0.0001%,0.000012%-about 0.0001%, 0.000014%-about 0.0001%, 0.000016%-about0.0001%, 0.000018%-about 0.0001%, 0.00002%-about 0.0001%, 0.00003%-about0.0001%, 0.00004%-about 0.0001%, 0.00005%-about 0.0001%, 0.00006%-about0.0001%, 0.00007%-about 0.0001%, 0.00008%-about 0.0001%, 0.00009%-about0.0001%, 0.000016%-about 0.00009%, 0.000018%-about 0.00009%,0.00002%-about 0.00009%, 0.00003%-about 0.00009%, 0.00004%-about0.00009%, 0.00005%-about 0.00009%, 0.00006%-about 0.00009%,0.00007%-about 0.00009%, or 0.00008%-about 0.00009% w/v for topicaladministration. In some embodiments, the suspensions include about0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%,0.00008%, 0.000081%, 0.000082%, 0.000083%, 0.000084%, 0.000085%,0.000086%, 0.000087%, 0.000088%, or 0.000089% w/v of the second activeagent.

The suspensions of the application may further comprise about 0.5 μM,about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM,about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM,about 8 μM, or about 9 μM of a second active agent, or apharmaceutically acceptable salt thereof, for example, nicotinic acid,nicotinamide, or vitamin K, or a combination thereof. In someembodiments, the concentration of the second active agent is about 1 μM.

The ophthalmic suspensions may contain various additives incorporatedordinarily, such as buffering agents (e.g., phosphate buffers, boratebuffers, citrate buffers, tartrate buffers, acetate buffers, aminoacids, sodium acetate, sodium citrate and the like), tonicity agents(e.g., saccharides such as sorbitol, glucose and mannitol, polyhydricalcohols such as glycerin, concentrated glycerin, PEG and propyleneglycol, salts such as sodium chloride), preservatives or antiseptics(e.g., benzalkonium chloride, benzatkonium chloride, P-oxybenzoates suchas methyl p-oxybenzoate or ethyl p-oxybenzoate, Benzyl alcohol,phenethyl alcohol, Sorbic acid or its salt, Thimerosal, Chlorobutanoland the like), solubilizing aids or particle stabilizing agents (e.g.,cyclodextrins and their derivative, water-soluble polymers such aspolyvinyl pyrrolidone, surfactants such as tyloxapol, polysorbates,poloxamer), pH modifiers (e.g., hydrochloric acid, acetic acid,phosphoric acid, sodium hydroxide, potassium hydroxide, ammoniumhydroxide and the like), thickening agents (e.g., HEC, hydroxypropylcellulose, methyl cellulose, HPMC, carboxymethyl cellulose and theirsalts), chelating agents (e.g., sodium edetate, sodium citrate,condensed sodium phosphate etc.), and second active agent stabilizers(e.g., EDTA, propyl gallate, and a combination thereof).

The ophthalmic suspension of the present application comprisespharmaceutical excipients chosen at or below concentrations optimal forophthalmic solution. The excipients of the present application include,for example, without being limiting, sodium phosphate monohydrate,glycerin, and benzalkonium chloride (BAK).

In some embodiments, the ophthalmic suspension comprises about0.01-about 0.5%, about 0.02-about 0.5%, about 0.04-about 0.5%, about0.06-about 0.5%, about 0.08-about 0.5%, about 0.08-about 0.4%, about0.08-about 0.3%, about 0.08-about 0.2%, about 0.08-about 0.18%, about0.08-about 0.16%, about 0.08-about 0.14%, or about 0.08-about 0.12%EDTA. In some embodiments, the ophthalmic suspension comprises about0.04%, about 0.06%, about 0.08%, about 0.1%, about 0.12, about 0.14%,about 0.16%, about 0.18%, or about 0.2% EDTA. In some embodiments, theophthalmic suspension comprises about 0.1% EDTA.

In some embodiments, the ophthalmic suspension comprises about0.001-about 0.5%, about 0.002-about 0.5%, about 0.005-about 0.5%, about0.01-about 0.5%, about 0.02-about 0.5%, about 0.03-about 0.5%, about0.04-about 0.5%, about 0.01-about 0.4%, about 0.01-about 0.3%, about0.01-about 0.2%, about 0.01-about 0.1%, about 0.01-about 0.08%, about0.01-about 0.06% propyl gallate. In some embodiments, the ophthalmicsuspension comprises about 0.01%, about 0.02%, about 0.03%, about 0.04%,about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09% propylgallate. In some embodiments, the ophthalmic suspension comprises about0.05% propyl gallate.

In some embodiments, the ophthalmic suspension comprises about 0.005%,about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%,about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 2.0%,about 3.0%, about 4.0%, or about 5.0% w/v of Formula I or II, or apharmaceutically acceptable salt thereof, for example, Compound-I, and asecond active agent, and may further comprise tromethamine (i.e., Tris).In some embodiments, the ophthalmic suspension comprises about 0.05%-1%Tris. In some embodiments, the ophthalmic suspension comprises about0.2%-1.0% Tris. In some embodiments, the ophthalmic suspension comprisesabout 0.4%-0.8% Tris. The tromethamine-based suspension of Compound-I orits free base and/or a second active agent may comprise additionalbuffers and excipients, for example, without being limiting, phosphatebuffer. The suspensions may further comprise one or more surfactant andemulsifier, for example, without being limiting, polysorbate 80 orequivalent excipients thereof; one or more nonionic liquid polymer ofthe alkyl aryl polyether alcohol type, for example, without beinglimiting tyloxapol; and/or one or more hydrophilic non-ionic surfactant,for example, without being limiting, poloxamer, such as poloxamer 407.

The present application provides suspensions of the agents of thepresent application formulated in the presence of excipients such as,without being limiting, Povidone, polysorbate 80 (PS80), polyethyleneglycol (PEG) 400, tyloxapol, poloxamer, glycerin, and BAK in a Trisbuffer.

In one embodiment the suspension of Compound-I or its free basecomprises about 0.1-0.5% phosphate buffer. In another embodiment thesuspension of Compound-I or its free base and the second active agentcomprises about 0.1-0.5% phosphate buffer. In some embodiments, the pHof the tromethamine-based suspension is between pH 4-7, for example, pH6.0. In some embodiments, the suspensions prepared in Tris furthercomprise about 0.5%-about 2% polysorbate 80; about 0.05-about 0.2%tyloxapol; and/or about 0.05%-about 0.4% poloxamer 407.

In some such embodiments, the suspensions of the application furthercomprise about 0.01-about 1%, or about 1-about 2.0% w/v glycerin. In aspecific embodiment, the suspensions comprise about 2% w/v glycerin.

In some embodiments, the suspensions of the application further compriseabout 0.001-about 0.005% w/v Benzalkonium chloride (BAK). The BAK amountmay be varied depending on any observed adverse effects. BAK may bedamaging to the cells on the ocular surface, and, therefore, the amountin the formulation may be varied to achieve an optimal level of ocularpenetration of Compound-I, without compromising the ocular cell layerintegrity and increased toxicity.

In some embodiments, the suspension optionally comprises buffers.Buffers when used, for example, can be sodium monophosphate basic,phosphoric acid and Tris buffer. Compound-I concentration in suspensionis about 0.005%-about 5.0% w/v. The suspension prepared withoutadditional buffer further comprises about 0.005% BAK and about 2%glycerin and with a pH 6.0. In another embodiment the suspensionprepared without additional buffer comprises about 1% polysorbate 80,about 0.1% tyloxapol, about 0.2% Poloxamer 407, about 0.005% BAK, about2.0% glycerin, and with a pH 6.0.

In suspensions prepared in phosphoric acid/Tris, the suspensioncomprises about 0.14% phosphoric acid, about 0.2% Tris base, about 1.0%polysorbate 80, about 0.005% BAK, about 2.0% glycerin and with a pH 6.0.In one embodiment, the suspension further comprises about 0.2%tyloxapol. The pH of the suspension varies between about pH 6.0 and 7.2.

The suspensions prepared in tromethamine (Tris) alone comprise about 1%polysorbate 80, about 0.1% tyloxapol, about 0.2% Poloxamer 407, about0.6% Tris, about 0.005% BAK, and about 2.0% glycerin with pH 6.0. Inanother embodiment, a suspension prepared in Tris comprises, about 1%Tris, about 0.45% NaCl, about 0.025% EDTA, about 0.2% HPMC, about 0.1%polysorbate 80, about 0.005% BAK, with a pH 6.0. In these suspensions 1N HCl and/or 1N NaOH are used for titration to appropriate pH.

The suspension of the application comprises about 0.005%, about 0.05%,about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%,about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 2.0%, about 3.0%,about 4.0%, or about 5.0% of an active agent of Formula I or II, or apharmaceutically acceptable salt thereof, for example, Compound-I, andabout 0.01%-about 0.05%, about 0.05-about 0.09%, or about 0.09-about0.2% w/v sodium phosphate monobasic monohydrate and/or about 0.3%-about1.0% of Tris. Alternatively, the suspension of the application comprisesabout 0.005%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about1.0%, about 2.0%, about 3.0%, about 4.0%, or about 5.0% of an activeagent of Formula I or II, or a pharmaceutically acceptable salt thereof,for example, Compound-I, about 0.005%, about 0.05%, about 0.1%, about0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about0.8%, about 0.9%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, orabout 5.0% of a second active agent, and about 0.01%-about 0.05%, about0.05-about 0.09%, or about 0.09-about 0.2% w/v sodium phosphatemonobasic monohydrate and/or about 0.3%-about 1.0% of Tris.

In a specific embodiment, the suspension comprises about 0.14% or about0.2% w/v Tris-buffer. In additional embodiments, suspensions areprepared in about 0.6% Tris or about 1.0% Tris. Other equivalent buffersystems well known in the art are also used in the suspensions of thepresent application. In one embodiment, the Formula II compound orCompound-I is formulated as about 0.4% active agent, about 5% CremophorRH40, about 2.0% glycerin, and about 0.005% BAK. In another embodiment,the Formula II compound or Compound-I is formulated as about 0.4% activeagent, about 0.005% to about 5.0% of the second active agent, about 5%Cremophor RH40, about 2.0% glycerin, and about 0.005% BAK.

In some embodiments, the suspension of the application has a pH value ofabout 4.0 to about 7.5 at or under about 40° C.

In some embodiments, the suspension of the application has a pH value ofabout 5.0 to about 7.0 at or under about 40° C.

For example, the suspension of the application has a pH value of about6.0 at or under about 40° C.

In some embodiments, the first active agent may be formulated as asolution according to the embodiments described herein, and the secondactive agent may be formulated as a suspension according to theembodiments described herein.

In other embodiments, the first active agent may be formulated as asuspension according to the embodiments described herein, and the secondactive agent may be formulated as a solution according to theembodiments described herein.

Concentration in Various Ocular Tissues—Delivered as an OphthalmicSuspension

In some embodiments, a suspension of Compound-I or its free baseprovides similar concentration of the first active agent at the centralchoroid and the central retina compared to the concentration of thefirst active agent delivered in Gel Drop form (discussed infra).

In some embodiments, Tris-based suspension of Compound-I or its freebase with or without a second active agent increases the bioavailabilityof the first active agent at the central choroid and the central retina,while reducing concentration at the cornea and preventing and/ortreating corneal disruptions and or diseases (e.g., corneal edema,ulceration, abnormalities, etc.). In some embodiments, topical deliveryof Compound-I or its free base formulated in Tris-base reduces cornealconcentration of Compound-I by about 5-10×, 10-20×, 20-30×, 30-40×, orabout 50-100× compared to the corneal concentration of equimolarCompound-I or its free base delivered as a Gel Drop.

The combined effects of decreasing corneal drug exposure so as to avoidpoor ocular tolerability while maintaining or increasing posteriorsegment bioavailability so as to increase inhibition of receptortyrosine kinase (RTK), for example, VEGFR, significantly increases thetherapeutic index and corresponding benefits to patients. The additionalprevention and/or treatment of disruptions to the anterior surface ofthe eye with a second active agent, such as an EGFR modulator (e.g.,activator), further improves the therapeutic index and correspondingbenefits to patients.

Once or twice daily administration of about 0.005%-about 5.0% w/vCompound-I suspension of the present application for 30-90 days or 4-6months is well tolerated in the eye.

Gel Drop

In some embodiments the ophthalmic composition or formulation of thepresent application is formulated as a Gel Drop. The Gel Dropformulation includes no more than about 0.05% of sodium phosphatemonobasic monohydrate to provide the required buffering capacity andfree-flowing, filterable formulations at about 0.005%-about 2.0%Compound-I and/or at about 0.005%-about 5% of a second active agentwithout the need for surfactant additives.

The Gel Drop formulation of the application comprises about 0.005% toabout 2.0% w/v of the first active agent of Formula I or II, or apharmaceutically acceptable salt thereof, for example, Compound-I. Theconcentration of Compound-I or its free base (Formula II) in the GelDrops may be about 0.005%-about 0.01%, about 0.01%-about 0.05%, about0.05%-about 0.1%, about 0.1%-about 0.2%, about 0.2%-about 0.3%, about0.3%-about 0.4%, about 0.4%-about 0.5%, about 0.5%-about 0.6%, about0.6%-about 0.7%, about 0.7%-about 0.8%, about 0.8%-about 0.9%, about0.9%-about 1.0%, or about 1.0%-about 2.0% w/v for topicaladministration. In some embodiments, the Gel Drops include about 0.005%,about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%,about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, or about 2%w/v of Compound-I or its free base (Formula II).

The Gel Drop formulation of the application may further comprise about0.00001%-about 5.0% w/v of the second active agent, or apharmaceutically acceptable salt thereof, for example, nicotinic acid,nicotinamide or vitamin K, or a combination thereof. The Gel Dropformulation of the application may further comprise about 0.00001%-about1.0%, about 0.00001%-about 0.1%, about 0.00001%-about 0.01%, about0.00001%-about 0.001%, about 0.00001%-about 0.0002%, or about0.00001%-about 0.0001% w/v of the second active agent, or apharmaceutically acceptable salt thereof, for example, nicotinic acid,nicotinamide or vitamin K, or a combination thereof. The concentrationof the second active agent in the Gel Drops may be about 0.00001%-about0.0001%, 0.000012%-about 0.0001%, 0.000014%-about 0.0001%,0.000016%-about 0.0001%, 0.000018%-about 0.0001%, 0.00002%-about0.0001%, 0.00003%-about 0.0001%, 0.00004%-about 0.0001%, 0.00005%-about0.0001%, 0.00006%-about 0.0001%, 0.00007%-about 0.0001%, 0.00008%-about0.0001%, 0.00009%-about 0.0001%, 0.000016%-about 0.00009%,0.000018%-about 0.00009%, 0.00002%-about 0.00009%, 0.00003%-about0.00009%, 0.00004%-about 0.00009%, 0.00005%-about 0.00009%,0.00006%-about 0.00009%, 0.00007%-about 0.00009%, or 0.00008%-about0.00009% w/v for topical administration. In some embodiments, the GelDrops include about 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%,0.00006%, 0.00007%, 0.00008%, 0.000081%, 0.000082%, 0.000083%,0.000084%, 0.000085%, 0.000086%, 0.000087%, 0.000088%, or 0.000089% w/vof the second active agent.

The Gel Drop formulation of the application may further comprise about0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7μM, about 8 μM, or about 9 μM of a second active agent, or apharmaceutically acceptable salt thereof, for example, nicotinic acid,nicotinamide, or vitamin K, or a combination thereof. In someembodiments, the concentration of the second active agent is about 1 μM.

In some embodiments, the Gel Drop ophthalmic compositions of the presentapplication include glycerin as a tonicity agent. Some embodiments ofthe application provide ophthalmic composition including mannitol. Theglycerin or mannitol content at an amount to prevent any changes in thesolubility of Compound-I, and at a level of about 2.0-about 2.5%,glycerin provides an osmolality of about 225-about 300 mOsm/kg dependingon the phosphate concentration. In additional embodiments, glycerin isabout 2% and phosphate is about 0.05% of the gel drop ophthalmiccomposition. The concentrations of glycerin and phosphate of the presentapplication is in an amount that the tonicity level of the ophthalmiccomposition is about 240 mOsm/kg.

The Gel Drop ophthalmic composition of the present application mayfurther include Benzalkonium Chloride (BAK). In some embodiments, theBAK content is about 0.005%, sufficient for preservation of theophthalmic composition against microbial contamination. In someembodiments of the present application, BAK is not required for use ofophthalmic composition in a sterile, single-use product.

In some embodiments, the Gel Drop ophthalmic formulation of Compound-Iincludes: about 0.005%-about 2.0% Compound-I or its free base, about0.05% sodium phosphate, about 2% glycerin as the tonicity adjustingagent, about 0.005% BAK as a preservative, water (purified, i.e.,distilled, or deionized) as a vehicle, and sodium hydroxide to adjust pHto 6.0. In one embodiment, no other excipients are added.

The Gel Drop of the application comprises about 0.005%-about 2.0% of theactive agent of Formula I or II, or a pharmaceutically acceptable saltthereof, for example, Compound-I, and about 0.01%-about 0.05%, about0.05-about 0.09%, or about 0.09-about 0.2% w/v sodium phosphatemonobasic monohydrate. In a specific embodiment, the Gel Drop comprisesabout 0.05%, about 0.05-0.2%, or about 0.2% w/v sodium phosphatemonobasic monohydrate buffer. Other equivalent buffer systems well knownin the art are also used in the Gel Drop of the present application. Inone embodiment, Compound-I or its free base is formulated as about0.4%-about 2.0% first active agent, about 5% Cremophor RH40, about 2.0%glycerin, and about 0.005% BAK.

In one embodiment, the Gel Drop of Compound-I includes about 0.3%-about2.0% (3-20 mg/mL) Compound-I, about 0.05%-about 0.2% Sodium Phosphate,and about 2% glycerin. The pH of the composition is between pH 5.0-7.0.

The Gel Drop ophthalmic formulation of Compound-I and a second activeagent includes: about 0.005%-about 2.0% Compound-I or its free base,about 0.00001%-about 5% (e.g., about 0.00001%-about 0.0002%, or about0.00001%-about 0.0001%) second active agent, about 0.05% sodiumphosphate, about 2% glycerin as the tonicity adjusting agent, about0.005% BAK as a preservative, water (purified, i.e., distilled, ordeionized) as a vehicle, and sodium hydroxide to adjust pH to 6.0. Inone embodiment, no other excipients are added.

The Gel Drop of the application comprises about 0.005%-about 2.0% of theactive agent of Formula I or II, or a pharmaceutically acceptable saltthereof, for example, Compound-I, about 0.00001%-about 5% (e.g., about0.00001%-about 0.0002%, or about 0.00001%-about 0.0001%) second activeagent, and about 0.01%-about 0.05%, about 0.05-about 0.09%, or about0.09-about 0.2% w/v sodium phosphate monobasic monohydrate. In aspecific embodiment, the Gel Drop comprises about 0.05%, about0.05-0.2%, or about 0.2% w/v sodium phosphate monobasic monohydratebuffer. Other equivalent buffer systems well known in the art are alsoused in the Gel Drop of the present application. In one embodiment,Compound-I or its free base is formulated as about 0.4%-about 2.0% firstactive agent, about 0.005%-about 5.0% second active agent, about 5%Cremophor RH40, about 2.0% glycerin, and about 0.005% BAK.

In one embodiment, the Gel Drop of Compound-I includes about 0.3%-about2.0% (3-20 mg/mL) Compound-I, about 0.005%-about 5.0% second activeagent, about 0.05%-about 0.2% Sodium Phosphate, and about 2% glycerin.The pH of the composition is between pH 5.0-7.0.

The present application provides Gel Drop of the agents (e.g., the firstactive agent and the second active agent) of the present applicationformulated in the presence of excipients such as, without being limitingexample, Povidone, polysorbate 80 (PS80), polyethylene glycol (PEG) 400,tyloxapol, poloxamer, glycerin, and BAK in a phosphate buffer.

Eye Drops

Disclosed herein is a formulation, comprising a first active agent,e.g., a compound of Formula I or II, and/or a second active agent, e.g.,nicotinic acid, nicotinamide, or vitamin K, or a combination thereof, aseye drops, a form of drug delivery that is pharmaceutically-acceptableto patients, convenient, safe, with an onset of action of severalminutes. A standard eye drop used in therapy according to U.S. federalregulatory practice is sterile, have a pH of about 6.0-7.4, and, if tobe used more than once, contains a preservative but has a limited shelflife after opening, usually one month. If the eye drops are packaged ina sterile, single use only unit-dose dispenser, the preservative can beomitted.

One method of eye drop formulation comprises the purest forms of thedisclosed compound of Formula I or II (e.g., greater than 99% purity)and/or of the second active agent, and the compound and/or the secondactive agent are mixed with buffer and tonicity adjusters, to adjust forphysiological pH and osmolarity. Examples of buffering agents tomaintain or adjust pH include, but are not limited to, acetate buffers,citrate buffers, phosphate buffers and borate buffers. Examples oftonicity adjustors are sodium chloride, mannitol and glycerin. In someembodiments, other pharmaceutically acceptable ingredients are alsoadded.

The formulated solution is then aliquoted into either a plurality ofdiscrete, sterile disposable cartridges each of which is suitable forunit dosing, or a single cartridge for unit dosing. Such a singledisposable cartridge is, for example, a conical or cylindrical specificvolume dispenser, with a container having side-walls squeezable in aradial direction to a longitudinal axis in order to dispense thecontainer contents therefrom at one end of the container.

The present application provides ophthalmic eye-dropsolutions/suspensions packaged in multi-dose form or single dose form,for example, as a plastic bottle with an eye-dropper. In multi-dose formformulations, preservatives are required to prevent microbialcontamination after opening of the container. Suitable preservativesinclude, but are not limited to: benzalkonium chloride, thimerosal,chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol,edetate disodium, sorbic acid, polyquarternium-1, or other agents knownto those skilled in the art, and all of which are contemplated for usein the present application. Such preservatives are typically employed ata level of from 0.001 to about 1.0% weight/volume.

Without wishing to be bound by theory, the formulation of the presentapplication in an eye drop provides a pulse entry of the drug. The routeby which Compound-I obtains access to the posterior segment is not bydirect diffusion through the cornea with subsequent diffusion throughthe aqueous humor, vitreous humor, retina and ultimately the choroid.Rather, the Compound-I compound achieves notable bioavailabilityposteriorly following topical instillation using a circumferential routearound, rather than through, the globe.

In certain clinical conditions, the eye drop solutions/suspensions canbe formulated with other pharmaceutical agents, in order to attenuatethe irritancy of the other ingredient and to facilitate clinicalresponse. Such agents include, but are not limited to, a vasoconstrictorsuch as phenylephrine, oxymetazoline, napthazoline or tetrahydrozoline;a mast-cell stabilizer such as olopatadine; an antihistamine such asazelastine; an antibiotic such as tetracycline; a steroidalanti-inflammatory drug such as betamethasone; a non-steroidalanti-inflammatory drug such as diclofenac; an immunomodulator such asimiquimod or interferons; and antiviral agents such as valaciclovir,cidofovir and trifluridine. The doses used for the above describedpurposes vary, but are in an effective amount to suppress discomfort,itch, irritation, or pain in the eye. When the compositions are dosedtopically, the “pharmaceutically effective amount” of a compound ofFormula I or II can generally be in a concentration range of from 0.05mg/mL to about 10 mg/mL and the “pharmaceutically effective amount” ofthe second active agent can generally be in a concentration range offrom 0.05 mg/mL to about 10 mg/mL, with 1 to 4 drops of the compositionadministered as a unit dose 1 to 4 times per day. The most common methodof ocular drug delivery is the instillation of drops into the cornea(i.e., “eye drops”).

A key requirement is that the formulation be sterile and produced in asterile environment. An ideal disclosed compound for use in ophthalmicsolutions/suspensions should be soluble and/or miscible in aqueous mediaat normal ocular pH and tonicity. Moreover, the disclosed compoundsshould be stable, non-toxic, long acting, and sufficiently potent tocounteract dilution of drug concentration by blinking and tearing.

Dosage Forms

The formulation of the present application may be suitable forophthalmic use. In one embodiment the formulation is a solution. Thesolution of the present application may be a clear, colorless, sterile,isotonic, buffered aqueous free-flowing liquid preparation. The drugproduct (e.g., the first active agent and/or the second active agent)has a pH of approximately 6.0 and may be stored at +5° C. The drugproduct may be provided in a container closure system consisting of asemi-transparent ophthalmic dispenser bottle with a dropper tip and cap.

In some embodiments, the clinical concentration of Compound-I ophthalmicsolution or suspension is equal to or less than about 0.1 mg/mL, equalto or less than about 0.2 mg/mL, about 0.2-about 1.0 mg/mL, about0.3-about 1.0 mg/mL, about 0.4-about 1.0 mg/mL, about 0.5-about 1.0mg/mL, about 0.6-about 1.0 mg/mL, about 0.7-about 1.0 mg/mL, about0.8-about 1.0 mg/mL, about 0.9-about 1.0 mg/mL, about 1.0-about 2.0mg/mL, about 2.0-about 3.0 mg/mL, about 3.0-about 4.0 mg/mL, about4.0-about 5.0 mg/mL, about 5.0-about 6.0 mg/mL, about 5.0-about 10.0mg/mL, about 10-about 20 mg/mL, about 20-about 30 mg/mL, about 30-about40 mg/mL, or about 40-about 50 mg/mL.

In other embodiments, the clinical concentrations of Compound-I and thesecond active agent ophthalmic solution or suspension are independentlyequal to or less than about 0.1 mg/mL, equal to or less than about 0.2mg/mL, about 0.2-about 1.0 mg/mL, about 0.3-about 1.0 mg/mL, about0.4-about 1.0 mg/mL, about 0.5-about 1.0 mg/mL, about 0.6-about 1.0mg/mL, about 0.7-about 1.0 mg/mL, about 0.8-about 1.0 mg/mL, about0.9-about 1.0 mg/mL, about 1.0-about 2.0 mg/mL, about 2.0-about 3.0mg/mL, about 3.0-about 4.0 mg/mL, about 4.0-about 5.0 mg/mL, about5.0-about 6.0 mg/mL, about 5.0-about 10.0 mg/mL, about 10-about 20mg/mL, about 20-about 30 mg/mL, about 30-about 40 mg/mL, or about40-about 50 mg/mL.

In one embodiment of the present application the strength of thecompound of Formula I or II is about 0.005%-about 5.0% (about 0.5-about50 mg/mL). A desired pharmacologic activity (or concentration) of theformulation of the present application against pathologic choroidal andretinal neovascularization is achieved following ocular administrationof formulations containing about 0.005%, about 0.05%, about 0.1%, about0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about0.8%, about 0.9%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, orabout 5.0% w/v of Compound-I. The present application provides that,following topical ocular administration with an optimal dose (forexample, between about 0.005% and about 5.0%) the pharmacologicallyactive concentration is achieved and maintained in the central choroidtarget tissue. In one embodiment the first active agent (Formula II orCompound-I) is formulated as about 0.005-about 5.0% w/v concentration,the second active agent (nicotinic acid, nicotinamide, or vitamin K, ora combination thereof) is formulated as about 0.005-about 5.0% w/vconcentration, and the combination is dosed once or twice a day per eyefor more than 60 consecutive days. The plasma concentrations observedfollowing topical administration are substantially below the levelexpected to produce systemic toxicity.

TABLE 2 In vitro Summary of pharmacodynamic properties for Compound-I InVitro Assay IC₅₀ = nM (ng/mL) Inhibition of recombinant VEGFR-2 tyrosinekinase 10.55 (6) using exogenous substrate Inhibition of recombinantFGFR-2 tyrosine kinase 8.79 (5) using exogenous substrate Inhibition ofrecombinant PDGFR tyrosine kinase 2636.67 (1500) using exogenoussubstrate Inhibition of recombinant EGFR tyrosine kinase 5853.40 (3330)using exogenous substrate Inhibition of recombinant IR tyrosine kinaseusing 10283.00 (5850) exogenous substrate Inhibition of VEGF-stimulatedVEGFR-2 5.27 (3) autophosphorylation in intact cells Inhibition ofVEGF-stimulated mitogenesis in 14.06 (8) HUVECs

In some embodiments, Compound-I exhibits potent inhibition of tyrosinekinase activity for several proangiogenic growth factor receptors, withIC₅₀ of less than about 100 nM (see Table 3). Compound-I also blocks thehigh-affinity VEGF receptors, e.g., VEGFR-1/Flt-1, but with lowerpotency (with IC₅₀ of about 122 nM (69.41 ng/mL)).

TABLE 3 In Vitro Inhibition of Tyrosine Kinases using a 10-pointTitration Curve (257 nM-5000 nM) for Compound-I Kinase IC₅₀ ₌ nM (ng/mL)for Compound-I AURKB (Aurora B) 207 (117.76) FGFR-1 8.50 (4.84) FGFR-23.08 (1.75) FGFR-3 33.9 (19.29) FGFR4 500 (284.45) FLT1 (VEGFR-1) 122(69.41) FLT3 419 (238.37) FLT4 (VEGFR-3) 54.2 (30.83) FYN 161 (91.59)KDR (VEGFR-2) 1.27 (0.72) PDGFRA (PDGFR alpha) 3120 (1774.97) PDGFRB(PDGFR beta) 1860 (1058.16) TEK (Tie2) 10.1 (5.75) RET 11.1 (6.31)

Although VEGFR inhibition appears to be essential for reducing vascularpermeability and preventing further neovascular growth, the simultaneousinhibition of VEGF signaling with inhibition of other growth factorsignaling pathways (e.g., PDGF and angiopoietins/Tie2) may be linked tounique therapeutic outcomes. The therapeutic outcomes of a broaderinhibition of signaling pathways may contribute to the regression ofnewly established pathologic vessels in the posterior segment of theeye.

In some embodiments, about 300 nM (about 170.67 ng/mL) of Compound-Iinhibits VEGFR-2 kinase function (see Table 4). Substantial blockade ofa similar set of proangiogenic growth factor receptors, includingFGFRs1-3, Tie-2, and EphB-4 are also observed. An unexpected finding isthat about 300 nM concentration of Compound-I inhibits the VEGFR-2kinase function, which falls within the typical range found in thecentral choroid and retina following five days of topical oculardelivery.

TABLE 4 In Vitro Inhibition of Tyrosine Kinases by 300 nM (170.67 ng/mL)Compound-I Kinase Mean % Inhibition at 300 nM Compound-I EPHB-4 87FGFR-1 96 FGFR-2 103 FGFR-3 (K650E variant) 104 FLT4 (VEGFR-3) 86 KDR(VEGFR-2) 104 RET 98 RET (Y791F mutation) 97 TEK (Tie2) 96

TABLE 5 In vitro Inhibition of Tyrosine Kinases by 1 μM (568.9 ng/mL)Compound-I Kinase Mean % Inhibition at 1 μM Compound-I ABL1 92 ABL1E255K 90 ABL1 G250E 89 ABL1 T315I 101 ABL1 Y253F 93 ACVR1B (ALK4) 98AURKB (Aurora B) 82 BRAF V599E 85 EPHA-1 81 EPHA-8 85 EPHB-1 83 EPHB-480 FGFR-1 98 FGFR-2 99 FGFR-3 96 FGFR-3 K650E 100 FGR 91 FLT-1 (VEGFR-1)86 FLT-4 (VEGFR-3) 95 KDR (VEGFR-2) 98 LCK 97 LYN A 81 LYN B 91 MAP4K4(HGK) 100 MAP4K5 (KHS1) 94 MAPK14 (p38 86 alpha) MINK1 100 PDGFRA T674I86 PTK6 (Brk) 88 RET 98 RET Y791F 94 SNF1LK2 82 SRC 91 SRC N1 83 TEK(Tie2) 99 YES1 98Overview of Drug Substance and Drug Product

Drug Product: Compound-I and/or second active agent ophthalmicformulations for clinical studies are manufactured in dosage strengthsbetween 0.05%-1.0% of Compound-I and about 0.00001%-about 5% (e.g.,about 0.00001%-about 0.0002%, or about 0.00001%-about 0.0001%) thesecond active agent. In some embodiments, Compound-I dosage in theformulation is 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.8%, or 1.0%. Insome embodiments, second active agent dosage in the formulation is0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.8%, 1.0%, 2.0%, 3.0%, 4.0% or5.0%. Compound-I and/or second active agent Ophthalmic Formulations(solutions or suspensions) are for daily, single use, topicaladministration to the eye in a clinical setting. In addition to thefirst and second active ingredients, in some embodiments the drugproduct may further contain about 0.005% BAK as a preservative, purifiedwater as vehicle, and is pH-adjusted with sodium hydroxide to pH 6.0.

Sodium Phosphate-Based Gel Drop

The ophthalmic benefits of Compound-I in a sodium phosphate-basedformulation (listed in Table 6) results from the self-gelling propertiesof the API in buffers, such as sodium phosphate. Spontaneous formationof self-forming, thixotropic gel of Compound-I from a clear solution isformed by increasing first active agent concentration in sodiumphosphate. Once the first active agent concentration in the phosphatebuffer reaches super-saturated state, insoluble particulates ofCompound-I are observed within the gel.

The current state of the art predicts that application of a gel withincreased viscosity to the surface of the eye would increase cornealresidence time. Increased corneal residence time in turn facilitatesocular drug absorption. As a result, the intraocular drug concentrationsof viscous gels would be increased in comparison to non-viscousformulations, such as water-like solutions. One way to increaseviscosity is to use various viscosity-enhancing excipients, e.g.,carboxymethylcellulose, which in effect achieves increased intraocularabsorption of different drug substances following topical ocularadministration. The present application provides a thixotropic gel ofCompound-I and/or a second active agent formed in the absence of anyviscosity-enhancing excipients. For example, when Compound-I orCompound-I and a second active agent are dissolved into a simple buffer,such as sodium phosphate, a thixotropic gel is formed. The thixotropicgel, which is formed without any viscosity-enhancing excipients, isformulated as a Gel Drop.

The present application provides dose-dependent and dose-frequencydependent delivery of Compound-I to the posterior segment eye tissues.

The Gel Drop formulations of the present application (listed in Table 6)differ among each other in several aspects, such as first activeconcentration, sodium phosphate concentration, presence or absence oftonicity (glycerin) or preservative (benzalkoniumchloride/BAK) agents,solubilizing surfactants (polysorbate 80, tyloxapol, and/or poloxamer),and pH.

Tromethamine-Based Suspension

The present application provides a suspension of Compound-I and/or asecond active agent in a tromethamine-based formulation. In someembodiments, the suspension of Compound-I and/or the second active agentin a tromethamine-based formulation has equal to or more than 95% of thefirst active drug substance in an insoluble form. This characteristic isdistinguishable from the soluble or semi-soluble state of Compound-I inthe Gel Drop (the Gel Drop (gel), which is not an entirely soluble stateas concentration of the first active agent increases) or in aCyclodextrin-based formulation. Tromethamine-based formulations ofCompound-I show increased turbidity with increasing first active agentconcentration. Administering a topical drop of Compound-I and/or secondactive agent suspension to the eye, which is a combination of solubleand insoluble first active agent components, are beneficial with respectto both safety/tolerability and efficacy.

The present application provides Compound-I in the tromethamine-basedsuspension, delivered at concentrations to the target tissues between10-1000× of the cellular IC₅₀ for the various pro-angiogenic RTKs. See,e.g., Table 7.

The corneal safety and tolerability of topical Compound-I is a directconsequence of the amount of soluble (as opposed to insoluble) firstactive agent applied to the corneal surface, and the resultant cornealtissue concentration. In some embodiments, subjects who receive topicalocular administration of the tromethamine-based suspension are able totolerate up to higher level of the first active agent concentration inthe formulation, as compared to equimolar formulations of the sodiumphosphate-based Gel Drop. The corneal safety and tolerability of topicalCompound-I is also a consequence of the administration of a secondactive agent, e.g., nicotinic acid, nicotinamide, or vitamin K, or acombination thereof, which is a modulator (e.g., activator) of EGFR thatprevents or treats corneal disruptions or diseases caused by inhibitionof EGFR.

TABLE 6 PK results with topical ocular formulation of Compound-I inSodium Phosphate-based Gel Drop MEAN 5D Mean SD Dose x [cho- [cho- [ret-[ret- Mean SD CD* Phos BAK Gly PS80 Tylox Polox Days per roid] roid]ina] ina] [AH] [AH] [plasma] mg/ml pH % % % % % % Osmo Dosing Day nM nMnM nM nM nM nM 6 5.9 0.05 0.005 2 1 0.1 0.2 270 4 3x 22.40 222 15.6 5 60.2 0.005 2 273 5 3x 1160 6 5.9 0.15 80 4 3x 823 50.9 12.5 6 6.1 0.050.005 2 1 0.1 248 5 3x 779 6 5.9 0.1 0.005 2 0.2 257 4 3x 768 55.4 8.434 6.0 2 0.005 2 1 0.2 5 3x 760 150   97.6 30.2 21.1 8.24 8.06 2 6.1 0.050.005 2 1 0.1 239 5 3x 660 5 6 0.2 0.005 2 1 0.2 288 5 3x 612 2 6.0 0.020.005 2 1 0.2 5 3x 599 225   92.9 28.6 13.8 3.04 4.46 4 6.0 0.2 0.005 25 3x 596 105   68.3 53.4 25 5.4 5.84 2 6 0.005 2 232 5 3x 589 <LLoQ 305.41 2 6 0.05 0.005 2 1 261 5 3x 559 126 25.5 4.15 1 6.1 0.005 2 230 53x 537 2 6.0 0.2 0.005 2 5 3x 532 238*   34.8 69.5 14.8 1.81 4.47 2 6.00.05 0.005 2 5 3x 528 106   113 42.7 32.2 11.3 5.79 2 6.0 0.1 0.005 2 53x 525 51.3 42.9 50.5 18.9 5.69 5.71 1 6.0 0.2 0.005 2 5 3x 519 44.490.8 20.8 20.6 4.16 3.97 2 6.1 0.15 34 4 3x 466 13.7 4.81 2 6.1 0.005 2229 5 3x 462 1 6.0 0.2 0.005 2 1 0.2% 5 3x 423 34.1 32.7 37.8 6.27 1.062.89 2 6 0.05 0.005 2 244 5 3x 422 121 27.8 5.92 1 6 0.05 0.005 2 5 3x398 81.6 101 30.1 16.4 6.08 4.45 2 5.9 0.05 0.005 2 233 5 3x 362 1 60.005 2 234 5 3x 359 17.1 2 6 0.2 0.005 2 1 281 5 3x 357 102 19.7 4.94 26.1 0.15 22 5 3x 356 2 6.0 0.05 0.005 2 1 0.2 5 3x 349 44.1 85.7 19.618.2 6.91 4.36 1 6 0.005 2 1 0.1 0.2 237 5 3x 339 2 6 0 2 0.005 2 257 53x 316 <LLoQ 29.5 5.26 1 6.0 0.05 0.005 2 0.01 0.2 5 3x 295 48.7 46.854.1 9.09 0.989 3.49 1 6 0.05 0.005 2 1 254 5 3x *CD: Compound 1

TABLE 7 PK results with topical ocular Compound-I in tromethamine-basedsuspension MEAN SD Mean SD Days Dose x [cho- [cho- [ret- [ret- Mean SD[plas- CD* Phos BAK Gly PS80 Tylox Polox Dos- per roid] roid] ina] ina][AH] [AH] ma] mg/mL pH % Tris % % % % % Osmo ing Day nM nM nM nM nM nMnM 10 6 0.14 1 0.005 2 414 5 3x 1520 410 5 6.0 0.14 ~0.20 0.005 2 1 0.25 3x 1190 551 210 85.2 195 190 13.3 4 6.0 0.14 ~0.20 0.005 2 1 0.2 5 3x1040 397 151 71.7 30.2 9.43 9.2 6 6 0.6 0.005 2 366 4 3x 928 68.4 8.31 45.0 0.14% ~0.20 0.005 2 5 3x 915 203 139 34.7 40.4 6.73 8.18 4 7.0 0.14%~0.20 0.005 2 5 3x 770 226 169 122 27.3 6.55 5.4 5 6 0.14 1 0.005 2 3805 3x 758 34.9 6 6 1 0.005 2 214 4 3x 701 22 5.68 4 6.0 0.14% ~0.20 0.0052 5 3x 680 217 129 57.8 55.3 39.5 10.2 5 6.0 0.14% ~0.20 0.005 2 5 3x574 91.1 113 28.4 50.5 9.51 7.87 2 6.0 0.14% ~0.20 0.005 2 1 0.2 5 3x456 55.4 79.8 64 14.1 0.535 4.99 2 6.1 0.6 0.005 2 1 0.1 0.2 321 5 3x416 2 6.0 0.14% ~0.20 0.005 2 5 3x 405 121 51.5 35.7 12.9 6.17 3.39 2 60.14 ~0.20 0.005 2 276 5 3x 352 <LLoQ 19.9 5.4 2 6 0.6 0.005 2 312 5 3x321 1 6.0 0.14% ~0.20 0.005 2 5 3x 286 64.6 31.5 36.4 15.7 4.32 2.61 16.0 0.14% ~0.20 0.005 2 1 0.2 5 3x 202 27.8 18.5 36.9 3.07 0.41 1.91*CD: Compound-I

The present application provides ocular bioavailability of Compound-I inthe posterior segment upon administration of a tromethamine-basedsuspension. The ocular bioavailability of Compound-I in the posteriorsegment is directly proportional to the total amount of drug,Compound-I, administered (insoluble plus soluble, see Table 7). Althoughthe insoluble drug particulates are not readily available to anteriorsegment tissues; the inherent and unique physicochemical properties ofCompound-I allow both insoluble and soluble components to gain entry toposterior segment tissues, such as the choroid and retina. Consequently,even higher drug concentrations than those achieved with Gel Dropformulations containing equivalent amounts of the first active agent areachieved with the tromethamine-based suspension. Thus,tromethamine-based suspension provide: a) improved corneal tolerabilityand b) maintained or increased bioavailability to the posterior segment,particularly to the choroid, the primary target tissue for treatingneovascular (wet) AMD. In addition, a second active agent, e.g.,nicotinic acid, nicotinamide, or vitamin K, or a combination thereof,which is a modulator (e.g., activator) of EGFR that prevents or treatscorneal disruptions or diseases caused by inhibition of EGFR, preventsand/or treats corneal disruptions potentially associated with theadministration of Compound-I, thereby increasing the therapeutic indexof Compound-I.

Cyclodextrin-Based Solution

Cyclodextrins, which are cyclic oligosaccharides made up of six to eightdextrose units (α-, β-, and γ-CDs) joined through one to four bonds, arewell-known for their ability to act as a solubilizing agent forrelatively insoluble drugs. See Stella & He, Cyclodextrins, Toxicol.Pathol., 36: 30-42 (2008).

In some embodiments, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD, also knownas KLEPTOSE® HPB) at equal to or more than 1:6 molar ratio orSulfobutylether-β-cyclodextrin (SBE-β-CD, also known as CAPTISOL®) atequal to or more than 1:2 ratio in the proposed clinical formulation,Compound-I or its free base and a second active agent OphthalmicSolution, provide solubility that meets clinical dose strengths of0.1-1.2% Compound-I.

In some embodiments, cyclodextrin-based solutions of Compound-I or itsfree base and/or a second active agent not only have improved solubilityof the first active agent into a uniform solution, but, upon topicalocular administration, also have a novel and previously unobservedcharacteristic of significantly increased therapeutic index of the firstactive agent at the posterior segment of the eye. The solutions ofCompound-I and/or the second active agent of the present applicationreduce anterior segment exposure of Compound-I, thereby increasing theconcentration of the first active in the solution and increasing thefrequency of its delivery in order to maintain high posterior segmentconcentrations. Both of these beneficial characteristics are related tothe known property of cyclodextrin to form hydrophilic complexes withhydrophobic drugs. See Stella & He, Cyclodextrins, Toxicol. Pathol., 36:30-42 (2008). The administration of the second active agent as acombination with the first active agent prevents corneal disruptions ordiseases caused by inhibition of EGFR by systemic diseases (e.g.,cancer, diabetes), eye diseases, or administration of the first activeagent, e.g., a compound of Formula I or II, thereby increasing thetherapeutic index of the first active agent, e.g., Compounds of FormulaI or II.

When formulated with Compound-I or its free base and/or a second activeagent, cyclodextrin can form a clear, colorless solution which exhibitswater-like viscosity. Following topical ocular administration,Compound-I/cyclodextrin complex has the appearance of beingpharmacologically inactive and metabolically inert. TheCompound-I/cyclodextrin complex confers corneal tolerability untilcyclodextrin spontaneously dissociates from the first active agent, thusmaking available high concentration of Compound-I at its intended siteof action in the posterior segment of the eye, e.g., choroid and retina.

In some embodiments, cyclodextrin-based solutions of Compound-I lowercorneal exposures of Compound-I compared to Gel Drop formulations atsimilar drug concentrations. The use of cyclodextrin-based solutions ofCompound-I provides about 10× reduction in corneal concentrations, ascompared to dosing with equimolar formulations of the Gel Drop. In someembodiments, after 20-30 days of topical ocular dosing of about0.2-2.0%, e.g., about 0.6%, Compound-I as a cyclodextrin-based solution,no untoward findings are attributed to test-article or vehicle. Thepresent application provides higher concentrations of Compound-I withinthe posterior segment target tissues, such as at the central choroid andthe central retina, when cyclodextrin-based solution of Compound-I istopically applied. In some embodiments, the combined effects ofdecreasing corneal drug exposure so as to avoid poor oculartolerability, while increasing posterior segment bioavailability so asto increase RTK inhibition, significantly increases the therapeuticindex and corresponding benefit(s) to treated subjects. In otherembodiments, the combined effects of decreasing corneal drug exposure soas to avoid poor ocular tolerability and preventing or treating ofcorneal disruptions or disease by the second active agent, whileincreasing posterior segment bioavailability so as to increase RTKinhibition can significantly increase the therapeutic index andcorresponding benefit(s) to treated subjects.

The present application provides expansion of the therapeutic window forboth suspension-based formulations (see Example 3) and the cyclodextrinformulations of Compound-I due to significantly reduced exposure (about10-100× or 1-2 log reduction). The reduced exposure improves cornealsafety/tolerability, which allows higher concentrations or frequency ofdosing of Compound-I to be administered topically. The higherconcentration enables Compound-I to achieve higher back of the eyetarget tissue concentration, which improves the therapeutic efficacy ofCompound-I.

In some embodiments, topical ocular dosing of ophthalmic gel drops isassociated with high corneal tissue exposure (≧100 uM) and correspondinguntoward observations in the anterior segment, such as discomfort,corneal and conjunctival inflammation, corneal epithelial erosion and/orthinning and degeneration. In contrast, repeated topical ocular dosingof Compound-I ophthalmic solution produces corneal exposure that areroughly 5 to 10-fold lower than an equimolar dose of ophthalmic geldrops, and are free of untoward clinical or histopathologic findings.Topical ocular dosing with Compound-I ophthalmic solution also achievesequal or higher target therapeutic exposure in the central choroid incomparison to an equimolar dose of the ophthalmic gel drop. Overall, thecombination of decreased corneal exposure and corresponding improvedocular tolerability, while simultaneously maintaining or promoting drugdelivery to the posterior segment target tissues, along with improvedphysicochemical stability, provides greater benefit to subjects comparedto the ophthalmic Gel Drop formulation.

1- to 5-Day PK Results with Topical Ocular Compound-I inCyclodextrin-Based Solutions

The present application provides ocular pharmacokinetics of variousformulations and dose regimens of Compound-I following topical ocularadministration. Three dosage strengths in nine (9) different topicalocular formulations of Compound-I are used for dosing either once perday (q.d.) or twice per day (b.i.d.) for 1, 2, 3, 4, or 5 consecutivedays. Subjects each receive about 30 μL bilateral topical ocular dose ofone of three (3) Compound-I formulations, or vehicle formulation, usinga positive displacement pipette.

The composition of each Compound-I formulation is described in Table 8A.All doses were administered within ±1 hour of the scheduled dose time.On day 1, Groups 1, 2, 4-6, 8, 10, 11, 13, 15, and 17 receive one dose(q.d.) for either one (1) or four (4) days. On days 1 through 4, Groups3, 7, 9, 12, 14, and 16 receive b.i.d. dosing approximately 8 hoursapart at 7:00 μM and 3:00 μM for four (4) days. Some subjects receiveb.i.d dosing of vehicle only formulations for five (5) consecutive days.

In some embodiments, ocular sampling is performed at about 0.5, about 1,about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23, orabout 24 hours post-dose relative to the day 1 dose. Aqueous humor,cornea, central and peripheral retina, and central and peripheralchoroid samples are collected to monitor effects of treatment. Aqueoushumor, cornea, central retina, and central choroid samples are assayed.

Table 8A-C lists 1- to 5-day PK results with topical ocular Compound-Iin Cyclodextrin-based solutions.

TABLE 8A Ocular Formulations Composition: 0.3% Compound-I (3 mg/mLCompound-I) 0.05% Sodium Phosphate, monobasic, monohydrate, USP 2.0%glycerin, USP pH 6 Physical Description: Clear and colorless, extremelyviscous Composition: 0.3% Compound-I (3 mg/mL Compound-I) 0.05% SodiumPhosphate, monobasic, monohydrate, USP 2.0% glycerin, USP pH 5.5Physical Description: Clear and colorless Composition: 0.4% Compound-I(4 mg/mL Compound-I) 7% Hydroxypropyl-β-cyclodextrin (HPβCD), 0.7%Sodium Chloride, USP, 0.005% Benzalkonium chloride (BAK), NF pH 7.0Physical Description: Clear and colorless, viscous Composition: 0.4%Compound-I (4 mg/mL Compound-I) 4% Hydroxypropyl-β-cyclodextrin (HPβCD),0.7% Sodium chloride, USP, 0.005% Benzalkonium chloride (BAK), NF pH 7.0Physical Description: Clear and colorless, viscous Composition: 0.4%Compound-I (4 mg/mL Compound-I) 4% Hydroxypropyl-β-cyclodextrin (HPβCD),0.7% Sodium Chloride, USP, 0.005% Benzalkonium chloride (BAK), NF pH 6Lot Number: BCL532-052(5) ALG-001 Physical Description: Clear andcolorless, extremely viscous Composition: 0.4% Compound-I (4 mg/mLCompound-I) 7% Hydroxypropyl-β-cyclodextrin (HPβCD), 1% Tromethamine,USP, 0.4% Sodium Chloride, USP, 0.005% Benzalkonium chloride (BAK), NF,pH 7.0 Physical Description: Clear and colorless Composition: 0.6%Compound-I (6 mg/mL Compound-I) 7% Hydroxypropyl-β-cyclodextrin (HPβCD),0.7% Sodium Chloride, USP, 0.005% Benzalkonium chloride (BAK), NF pH 7.0Physical Description: Clear and colorless, viscous Composition: 0.6%Compound-I (6 mg/mL Compound-I) 7% Hydroxypropyl-β-cyclodextrin (HPβCD),0.7% Sodium Chloride, USP, 0.005% Benzalkonium chloride (BAK), NF, pH6.0 Physical Description: Clear and colorless, viscous Composition: 0.4%Compound-I (4 mg/mL Compound-I) 5% Cremophor RH40, 2.0% glycerin, USP,0.005% Benzalkonium chloride (BAK), NF, pH 6.0 Physical Description:Clear and colorless

Table 8B lists average Compound-I concentrations in aqueous humor,retina, choroid, and cornea (LLOQ: Lower Limit of Quantitation; the LLOQis the lowest analyte concentration that can be quantified withacceptable precision and accuracy).

TABLE 8B Average Concentration of Compound-I (μM) Time Aqueous CentralPeripheral Central Peripheral Group Point Humor Retina Retina ChoroidChoroid Cornea 1 0.5 hr 0.00162 0.0404 0.0291 <LLOQ <LLOQ 57.6 1 hr0.00206 <LLOQ 0.0548 <LLOQ *0.0856 33.7 2 hr 0.0103 0.0368 0.0779 <LLOQ0.0575 44.5 8 hr 0.0128 0.0340 0.0356 <LLOQ 0.134 29.2 24 hr 0.00303<LLOQ 0.0151 <LLOQ 0.0880 6.94 2 1 hr 0.00996 0.0363 0.0961 *0.207 0.737 112 8 hr 0.0165 0.0380 0.0508 *0.237  0.687 32.7 24 hr 0.00336<LLOQ 0.380 *0.205  1.14 29.2 3 1 hr 0.0142 0.0407 0.108 0.255 0.765 15124 hr 0.00774 0.0292 0.0597 0.283 0.892 82.1 4 1 hr 0.00996 0.04310.0883 0.196 0.629 78.7 5 0.5 hr <LLOQ <LLOQ 0.0227 <LLOQ <LLOQ 21.0 1hr 0.00108 0.0211 0.0253 <LLOQ 0.0473 16.9 2 hr 0.00862 0.0354 0.0253<LLOQ 0.0509 28.1 4 hr 0.00911 0.0299 0.0312 <LLOQ 0.0775 14.1 8 hr0.00667 0.0304 0.0333 <LLOQ 0.0874 7.74 24 hr 0.00228 <LLOQ *0.0103<LLOQ 0.116 2.43 6 1 hr 0.00323 0.0463 0.0634 0.319 0.311 21.8 8 hr0.00742 0.0537 0.0349 <LLOQ 0.257 9.10 24 hr 0.00122 0.0241 0.0533 <LLOQ0.343 2.33 7 1 hr 0.00648 0.0469 0.0819 0.514 0.744 35.3 24 hr 0.002600.0313 0.0293 0.439 0.653 13.0 8 1 hr 0.00978 0.0490 0.0497 0.367 0.79763.2 24 hr 0.00483 0.0193 0.0177 0.218 1.20 37.4 9 1 hr 0.0246 0.0633N/A 0.456 N/A 237 10 1 hr 0.00867 0.0667 N/A 0.251 N/A 93.9 AH LLOQ =0.000903 μM Central Retina LLOQ = 0.0181 μM Peripheral Retina LLOQ =0.00873 μM (Grps 1-8); LLOQ = 0.00898 μM (Grps 12-16) Central ChoroidLLOQ = 0.175 μM Peripheral Choroid LLOQ = 0.0349 μM (Grps 1-8); LLOQ =0.0359 μM (Grp 12-16) Cornea LLOQ = 0.0181 μM (Grp 1-5); LLOQ = 0.0453μM (Grp 6-8, 10-13, 15, 16A17); LLOQ = 0.0873 μM (Grp 4, 9, 16B) N/A =Not Applicable; Samples not assayed per study protocol. *Average basedon n = 1.

Table 8C lists the summary of average ocular tissue concentrations ofCompound-I in aqueous humor, central and peripheral retina, central andperipheral choroid, and cornea for Groups 1 through 10. Any values <LLOQwere excluded from statistical calculations. When all values are <LLOQfor a given time point, <LLOQ are reported as the average.

TABLE 8C Average Concentration of Compound-I (μM) Time Aqueous CentralPeripheral Central Peripheral Group Point Humor Retina Retina ChoroidChoroid Cornea 11 1 hr 0.00284 0.0397 N/A 0.193 N/A 20.7 24 hr  *0.00205*0.0185 N/A *0.179 N/A 0.487 12 1 hr 0.00651 0.0521  0.0842 0.528 0.56027.4 13 1 hr 0.0102 0.0934 N/A 0.372 N/A 123 24 hr  0.00518 0.0246 N/A0.319 N/A 39.1 14 1 hr 0.0209 0.0817 0.151 7.19 1.00  236 15 1 hr 0.01140.0527 N/A 0.319 N/A 82.9 16 1 hr 0.0179 0.0480 0.169 0.495 0.868 169 171 hr 0.00445 0.0468 N/A 0.297 N/A 32.0 AH LLOQ = 0.000903 μM CentralRetina LLOQ = 0.0181 μM Peripheral Retina LLOQ = 0.00873 μM (Grps 1-8);LLOQ = 0.00898 μM (Grps 12-16) Central Choroid LLOQ = 0.175 μMPeripheral Choroid LLOQ = 0.0349 μM (Grps 1-8); LLOQ = 0.0359 μM (Grp12-16) Cornea LLOQ = 0.0181 μM (Grp 1-5); LLOQ = 0.0453 μM (Grp 6-8,10-13, 15, 16A17); LLOQ = 0.0873 μM (Grp 4, 9, 16B) N/A = NotApplicable; Samples not assayed per study protocol. *Average based on n= 1.5-Day PK Results with Topical Ocular Compound-I in Cyclodextrin-BasedSolutions

The present application provides ocular pharmacokinetics of various doseregimens of topical ocular solutions of Compound-I containinghydroxypropyl-β-cyclodextrin (“HDβCD”) following ocular doseadministration. Different topical ocular solutions of Compound-I areadministered either once per day (q.d.) or twice per day (b.i.d.) foreither 4 or 5 consecutive days. Subjects each receive a 30 μL bilateraltopical ocular dose of one of four Compound-I dosage strengths.

All doses were administered with ±1 hour of the scheduled dose time,except some subjects receiving on day 1. Ocular sampling afteradministration of Compound-I is performed one hour following the firstdaily dose on day 5 for subjects, except in a few, where ocular samplingis performed 24 hours after the first daily dose on day 4.

Aqueous humor, cornea, central and peripheral retina, and central andperipheral choroid samples are collected. Cornea, central retina, andcentral choroid samples are assayed; aqueous humor, peripheral retina,and peripheral choroid samples are not assayed.

Table 9 (A-B) lists 5-day PK results with topical ocular Compound-I incyclodextrin-based solutions.

TABLE 9A Ocular Formulations Formulation Composition: 0.4% Compound-I(as free base) 1 (A) 7.15% Hydroxypropyl-β-cyclodextrin 0.7% Sodiumchloride pH 6.5 Physical Clear and colorless Description: FormulationComposition: 0.1% Compound-I (as free base) 2 (B) 1.79%Hydroxypropyl-β-cyclodextrin 0.85% Sodium chloride pH 6.5 Physical Clearand colorless Description: Formulation Composition: 0.2% Compound-I (asfree base) 3 (C) 3.57% Hydroxypropyl-β-cyclodextrin 0.8% Sodium chloridepH 6.5 Physical Clear and colorless Description: FormulationComposition: 0.6% Compound-I (as free base) 4 (D) 10.72%Hydroxypropyl-β-cyclodextrin 0.6% Sodium chloride pH 6.5 Physical Clearand colorless Description: Formulation Composition: 0.4% Compound-I (asfree base) 5 (E) 8.41% Hydroxypropyl-β-cyclodextrin 0.65% Sodiumchloride pH 6.5 Physical Clear and colorless Description: FormulationComposition: 0.4% Compound-I (as free base) 6 (F) 10.51%Hydroxypropyl-β-cyclodextrin 0.65% Sodium chloride pH 6.5 Physical Clearand colorless Description: Formulation Composition: 0.4% Compound-I (asfree base) 7 (G) 10.51% Hydroxypropyl-β-cyclodextrin 0.15% Sodiumchloride 1.0% Tromethamine (Tris) pH 6.5 Physical Clear and colorlessDescription: Formulation Composition: 0.1% Compound-I (as free base) 8(H) 2.63% Hydroxypropyl-β-cyclodextrin 0.8% Sodium chloride pH 6.5Physical Clear and colorless Description: Formulation Composition: 0.6%Compound-I (as free base) 9 (I) 15.77% Hydroxypropyl-β-cyclodextrin0.37% Sodium chloride pH 6.5 Physical Clear and colorless Description:

Table 9B lists a summary of average ocular tissue concentrations ofCompound-I in central retina, central choroid, and cornea. Any values<LLOQ were excluded from statistical calculations. When all values were<LLOQ for a given time point, <LLOQ was reported as the average.

TABLE 9B Average Compound-I concentrations in retina, choroid, andcornea. Average Concentration (μM) Time Central Central Group PointRetina Choroid Cornea 1 1 hr 0.0670 0.308 35.1 2 1 hr 0.0636 0.329 21.83 1 hr 0.0579 0.313 18.2 4 1 hr 0.0481 0.203 12.9  5a 1 hr 0.0403 *0.19912.8  5b 24 hr  <LLOQ 0.194 0.772  6a 1 hr 0.0469 0.309 10.6  6b 24 hr <LLOQ *0.218 0.371 7 1 hr 0.0332 <LLOQ 7.60 8 1 hr 0.0376 *0.210 5.41 91 hr 0.0261 *0.287 8.53 10  1 hr 0.0534 0.264 16.7 11  1 hr 0.0418 0.37129.6 12  1 hr 0.0464 0.210 16.3 Central Retina LLOQ = 0.0218 μM CentralChoroid LLOQ = 0.174 μM Cornea LLOQ = 0.0174 μM *Average based on n = 1Concentrations of Compound-I (in μM) in Various Ocular Fluids andTissues

In some embodiments, concentration of the first active agent in varioustissues and fluids of the eye is measured upon topical ocularadministration of a solution of about 0.4% (about 4 mg/mL) Compound-Iand cyclodextrin. Average concentration of Compound-I is measured in thecentral choroid, central retina, aqueous humor, and cornea. Compound-Iis in a solution (0.4% or 4 mg/mL) with 8.41% KLEPTOSE® and 0.142%phosphate buffer; 8.9% KLEPTOSE® HPB and 0.142% phosphate; 4.88%CAPTISOL® and 0.142% phosphate; or 4.88% CAPTISOL® and 0.122% phosphate.See Table 10A-B.

In some embodiments, upon topical ocular administration of a solution ofabout 0.4% (about 4 mg/mL) Compound-I and cyclodextrin, the centralchoroid concentration of Compound-I is between about 0.2 μM and about0.8 μM. The central retina concentration of Compound-I is between about0.05 μM-about 0.15 μM. In some embodiments, upon topical ocularadministration of a solution of about 0.4% (about 4 mg/mL) Compound-Iand cyclodextrin, the aqueous humor concentration of Compound-I isbetween about 0.003 μM-about 0.008 μM. And the corneal concentration ofCompound-I is about 6.0 μM-about 40 μM. KLEPTOSE® HPB or CAPTISOL® isused in the solution of Compound-I administered topically to the eye.

In some embodiments, mean Compound-I ocular tissue concentrationsfollowing twice daily topical dosing with 0.3% Compound-I ophthalmic geldrop formulations with and without benzylalkonium chloride is highest inthe cornea with between about 200 μM-about 350 μM in the cornea, betweenabout 2.0 μM-about 5.0 μM in the peripheral choroid, between about 0.2μM-about 0.7 μM in the central choroid, between about 0.05 μM-about 0.5μM in the peripheral retina, and between about 0.01 μM-about 0.05 μM inthe aqueous humor.

In some embodiments, Tris-based suspension formulations of Compound-I iswell tolerated, without any corneal findings, and only with a fewsporadic incidences of mild conjunctivitis. In some embodiments, meanCompound-I ocular tissue concentrations, assessed at 1 hour±15 minutesafter the first daily topical ocular dose on day 30 for the twice dailytopical dosing with 0.3% Compound-I Tris-based suspensions with andwithout benzylalkonium chloride, are highest in the cornea, for example,between about 2.00 μM-about 4.0 μM. The peripheral choroid concentrationfrom the same dose is between about 0.7 μM-about 1.5 μM; the centralchoroid concentration is between about 0.3 μM-about 0.4 μM; theperipheral retina concentration is between about 0.08 μM-about 0.09 μM);central retina concentration is between about 0.04 μM-about 0.07 μM; andaqueous humor concentration is about 0.001 μM-about 0.002 μM.

The present application provides Cyclodextrin-based solutions (e.g.,solutions comprising hydroxypropyl-beta-cyclodextrin (HP-β-CD, KLEPTOSE®HPB)) of Compound-I that were well tolerated when administered topicallyfor up to 30 days, twice daily at about 0.1% Compound-I (in a solutionwith about 2.0%-about 2.5% HP-β-CD), twice daily at about 0.2%Compound-I (in a solution with about 4.0%-about 4.5% HP-β-CD), once ortwice daily at about 0.4% Compound-I (in a solution with about8.0%-about 8.5% HP-β-CD), and once or twice daily at about 0.6%Compound-I (in solution with up to about 14% HP-β-CD) in subjects.Moreover, in additional embodiments, cyclodextrin-based solutions ofabout 0.4% w/v Compound-I in KLEPTOSE® HPB, KLEPTOSE® HP, or CAPTISOL®are well-tolerated when dosed twice daily for up to 24 days.

The present application provides dose-limiting corneal toxicity observedwith Compound-I ophthalmic Gel Drop formulations. In some embodiments,ophthalmic Gel Drop renders about five-fold to about fifteen-fold highercorneal concentrations of Compound-I compared to cyclodextrin basedsolution, and about fifty-fold to about hundred-fold higher cornealconcentrations of Compound-I compared to Tris-based suspensions.Compound-I Tris-based suspensions and cyclodextrin-based solutions ofthe present application are well tolerated with no evidence of overtocular toxicity. In some embodiments, once or twice daily administrationfor at least 30 days of about 0.005% to about 5.0% w/v of acyclodextrin-based solution or a Tris-based suspension of Compound-I iswell tolerated in subjects. The present application provides highestcentral choroid concentrations of Compound-I using Cyclodextrin-basedsolutions compared to equimolar doses of the gels and/or Tris-basedformulations.

TABLE 10A Average concentration of Compound-I in μM in various ocularfluids and tissues Group Central Choroid Central Retina Aqueous HumorCornea 8 0.769 0.124 0.00656 12.3 9 0.259 0.0741 0.00313 8.05 10 0.2120.0531 0.00184 6.49 11 0.345 0.101 0.00403 30.0 Values <LLOQ wereexcluded from statistical calculations. Choroid LLOQ = 0.184 μM RetinaLLOQ = 0.0229 μM AH LLOQ = 0.000918 μM Cornea LLOQ = 0.0918 μM

TABLE 10B Study Design Total Dose Number Total Dose Number Daily Dose*Conc.* Volume of Doses Volume of Male Group (mg/day) (% w/v) (μL/dose)per Day (μL/day) Animals Group 8. Compound-I 0.48 0.4 30/eye 2 120 2 in8.41% KLEPTOSE ® HPB**, 0.142% phosphate Group 9. Compound-I 0.48 0.430/eye 2 120 2 in 8.90% KLEPTOSE ® HPB, 0.142% phosphate Group 10.Compound- 0.48 0.4 30/eye 2 120 2 I in 4.88% CAPTISOL ®***, 0.142%phosphate Group 11. Compound- 0.48 0.4 30/eye 2 120 2 I in 4.88%CAPTISOL ®, 0.122% phosphate *Total daily dose and concentration areexpressed as free base equivalent of Compound-I (Formula II).**Hydroxypropyl-β-cyclodextrin (HPβCD) from Roquette. ***CAPTISOL ® is apolyanionic β-cyclodextrin derivative with a sodium sulfonate saltseparated from the lipophilic cavity by a butyl ether spacer group, orsulfobutylether (SBE).

Table 11 shows the corneal and central choroidal concentrations ofCompound-I formulations.

TABLE 11 Central Com- Dosing Cornea Choroid pound- Formulation Frequency[Compound [Compound I w/v % Type & Duration I] (μM) I] (μM) 0.3%Ophthalmic Twice Daily 236.00 0.340 Gel Drop 29 days 0.3% Tris TwiceDaily 2.69 0.319 Suspension 30 days 0.4% Ophthalmic Twice Daily 6.490.212 Solution 24 days (CAPTISOL ®) 0.4% Ophthalmic Twice Daily 8.050.259 Solution 24 days (KLEPTOSE ® HP) 0.4% Ophthalmic Twice Daily 12.300.769 Solution 24 days (KLEPTOSE ® HPB)Phase I Protocol for Dose-Escalation Study in Patients with NeovascularAMD

The present application provides a Phase I study involving atwelve-week, open-label, dose-escalating, multi-center trial to evaluatethe safety, tolerability, and pharmacokinetics following topical ocularadministration of Compound-I in patients with neovascular age-relatedmacular degeneration (AMD). Up to 60 patients total are treated one totwo times daily with topical ocular dosing of Compound-I ophthalmicsolution for three months, where three dose-escalating monotherapy armsand one adjunct therapy arm using a single intravitreal injection ofLUCENTIS® plus the maximally-tolerated monotherapy dose are planned (15patients per treatment arm). Patients that meet pre-specified vision andCNV lesion criteria confirmed by an independent reading center areallowed to simultaneously discontinue topical ocular dosing and receivetreatment with standard-of-care.

The present application provides 3 dosage strengths, ranging from 0.1%to 1.0% (w/v) (as Compound-I) ophthalmic solution for clinical studies.The strengths are about 0.1%, about 0.3%, about 0.6%, and about 1.0%(w/v) Compound-I HCl.

Formulation Preparation

Non-limiting examples of formulations of the present application areoutlined in Table 12.

TABLE 12 Overview of product compositions tested in product screeningstudies Cyclodextrin Type and Cyclodextrin Compound-I Buffer type RatioRange^(a) Conc. Range Conc. pH and level HPβCD^(b)  6.3 to 18.9% 0.6% 7None and Tris 1:4, 1:8, 1:10, 1:12 HPβCD 1.58 to 15.6% 0.1 and 0.6% 6None and Tris 1:6, 1:8, 1:10 HPβCD 1.58 to 2.63% 0.1% 6.5, 7  None andTris 1:6, 1:10 SBECD 0.81 to 19.5% 0.1 and 0.6% 6 Phosphate 1:2, 1:3,1:4, 1:6, 1:8, 1:10 SBECD^(c) 1:3 0.1, 0.4% 5.5, 6.5 Phosphate, TrisHPβCD 1:8 0.1, 0.4% 5.5, 6.5 Phosphate, Tris ^(a)Molar ratio ofCompound-I: cyclodextrin. ^(b)KLEPTOSE ® HPB ^(c)CAPTISOL ®EGFR Tyrosine Phosphorylation Assay in Cells to Determine EGFR Activityof Compounds of Formula I or II

An EGFR tyrosine assay in corneal epithelial cells is run to determinewhether higher concentrations of EGF can overcome inhibition of EGFRkinase activity. Cells are serum starved and then are pre-treated withdifferent concentrations of a compound of Formula I or II, e.g.,Compound-I or a control for followed by treatment with EGF. Cells arethen harvested and immunoblotted for determination of phosphorylatedEGFR and total EGFR concentration which was used to determine receptoractivity (IC₅₀).

EGFR Tyrosine Phosphorylation Assay in Cells to Determine EGFR Activityof a Combination of Compounds of Formula I or II, and a Second ActiveAgent

An EGFR tyrosine assay in corneal epithelial cells is run to determineif Vitamin K or nicotinic acid/nicotinamide can overcome inhibition ofEGFR and if there is an increase in EGFR activity. Cells are serumstarved and then are pre-treated with a different concentration of acompound of Formula I or II, e.g., Compound-I and saturatingconcentrations of vitamin K or nicotinic acid/nicotinamide followed bytreatment with EGF. Cells are harvested cells and immunoblotted fordetermination of phosphorylated EGFR (tyrosine 1068 and tyrosine 1045)and total EGFR concentration which was used to determine receptoractivity (IC₅₀).

Determination of Effects of Varying Concentrations of Compounds ofFormula I or II and EGF on Cell Migration/Proliferation in Cells (InVitro Wound Healing)

Determination of cell migration/proliferation in corneal epithelialcells is run to determine cell migration/proliferation in the presenceof varying concentrations of a compound of Formula I or II and EGF.Cells are plated with silicone plugs. The cells are then serum starvedand pre-treated with varying concentrations of a compound of Formula Ior II or a control. The silicone plugs are then removed to create theacellular area the cells are treated with EGF. Cell migration isquantified from micrographs.

Determination Cell Migration/Proliferation in Cells Treated withCompounds of Formula I or II and/or a Second Active Agent

Determination of cell migration/proliferation in corneal epithelialcells is run to determine cell migration/proliferation in the presenceof varying concentrations of a compound of Formula I or II, vitamin K,or nicotinic acid/nicotinamide. Cells are plated with silicone plugs.The cells are then serum starved and pre-treated with varyingconcentrations of a compound of Formula I or II or a control. The plugsare removed and the cells are treated with 1) EGF, 2) EGF and vitamin Kor 3) EGF and nicotinic acid/nicotinamide. Cell migration is quantifiedfrom micrographs.

Determination of Effects of Compounds of Formula I or II on Basal andEGF-Mediated Corneal Wound Healing In Vivo

Determination of the effects of a compound of Formula I or II on basaland ligand stimulated rates of corneal wound healing was determined inmice. Corneas of C57/Bl mice are wounded and then pre-treated with acompound of Formula I or II followed by addition of EGF. Wound size ismonitored by fluorescein staining and fluorescent photography andquantified.

Determination of Effects of Compounds of Formula I or II and a SecondActive Agent on Basal and EGF-Mediated Corneal Wound Healing In Vivo

Determination of the effects of a compound of Formula I or II and/or asecond active agent on corneal wound healing was determined in mice.Corneas of C57/Bl mice are wounded and then pre-treated with a compoundof Formula I or II, followed by addition of EGF, vitamin K, or nicotinicacid/nicotinamide. Wound size and closure are monitored.

Doses of Treatment

The formulation of the present application is effective in treating(i.e., lesion stabilization or regression) or preventing choroidal andretinal neovascularization (NV) in the eye of a mammalian subject. TheCompound-I of the present application, at a specific dose, inhibits areceptor tyrosine kinase and the second active agent at a specific dose,inhibits an ErbB receptor tyrosine kinase. In some embodiments, theCompound-I formulation, at a specific dose, inhibits receptor tyrosinekinase including, VEGFR, FGFRs, Tie2, and EphB-4. The inhibition ofseveral RTKs by the formulation of the present application, at aspecific dose, simultaneously has a synergistic effect, and is effectivein the treatment or regression of NV in the posterior segment of theeye. In some embodiment, the second active agent modulates (e.g.,activates) directly or indirectly an ErB receptor tyrosine kinaseincluding, EGFR, HER2, HER3 and Erb4. The activation of several ErBreceptor tyrosine kinases by the formulation of the present application,at a specific dose, simultaneously may have a synergistic effect, and iseffective in the prevention or treatment of corneal disruptions ordiseases.

In one embodiment, the present application provides a method of treating(i.e., lesion stabilization or regression) or preventing choroidal andretinal neovascularization (NV) in the eye by administering to thesubject in need thereof a therapeutically effective amount of a compoundof Formula I or II, or a pharmaceutically acceptable salt thereof, and asecond active agent or a pharmaceutically acceptable salt thereof,wherein a compound of Formula I or II, or a pharmaceutically acceptablesalt thereof, and the second active agent, or a pharmaceuticallyacceptable salt thereof, are administered simultaneously. Alternatively,a compound of Formula I or II, or a pharmaceutically acceptable saltthereof, is administered prior to administration of a second activeagent, or a pharmaceutically acceptable salt thereof. In anotherembodiment, a compound of Formula I or II, or a pharmaceuticallyacceptable salt thereof, is administered after administration of asecond active agent, or a pharmaceutically acceptable salt thereof. Inanother embodiment, the present application provides a method oftreating (i.e., lesion stabilization or regression) or preventingchoroidal and retinal neovascularization (NV) in the eye byadministering to the subject in need thereof a therapeutically effectiveamount of a compound of Formula I or II, or a pharmaceuticallyacceptable salt thereof, prior to administering a therapeuticallyeffective dose of a formulation described herein.

In another embodiment, the present application provides a method oftreating (i.e., lesion stabilization or regression) or preventingchoroidal and retinal neovascularization (NV) in the eye and preventingor treating a corneal disruption or disease by administering to thesubject in need thereof a therapeutically effective amount of a compoundof Formula I or II, or a pharmaceutically acceptable salt thereof, and asecond active agent or a pharmaceutically acceptable salt thereof,wherein a compound of Formula I or II, or a pharmaceutically acceptablesalt thereof, and the second active agent, or a pharmaceuticallyacceptable salt thereof, are administered simultaneously. Alternatively,a compound of Formula I or II, or a pharmaceutically acceptable saltthereof, is administered prior to administration of a second activeagent, or a pharmaceutically acceptable salt thereof. In anotherembodiment, a compound of Formula I or II, or a pharmaceuticallyacceptable salt thereof, is administered after administration of asecond active agent, or a pharmaceutically acceptable salt thereof. Inanother embodiment, the present application provides a method oftreating (i.e., lesion stabilization or regression) or preventingchoroidal and retinal neovascularization (NV) in the eye and preventingor treating a corneal disruption or disease by administering to thesubject in need thereof a therapeutically effective amount of a compoundof Formula I or II, or a pharmaceutically acceptable salt thereof, priorto administering a therapeutically effective dose of a formulationdescribed herein.

In a further embodiment, the formulation of the present application iseffective in treating NV and treating and/or preventing cornealdisruption or disease caused by systemic disease, an eye disease oradministration of a compound of Formula I or II when administered one,two, three, and four times daily by topical ocular delivery of about0.005%-about 5.0% (about 0.05-about 50 mg/mL) of Compound-I and a secondactive agent. The formulation of Compound-I or its free base (FormulaII) and a second active agent, for the treatment or regression of NV andthe treatment and/or regression of corneal disruption or disease, is asolution comprising a second active agent, a compound of Formula I orII, and cyclodextrin or in a suspension comprising Tris. The solution orsuspension when delivered to a subject exposed to atmospheric oxygen toinduce oxygen induced retinopathy (OIR) or NV, for example, is able toeffectively reduce the mean area of pre-retinal NV per retina withlittle or no corneal disruption or disease. The prevention or treatmentof NV by Compound-I formulation and/or suspension is achieved viainhibition of several receptor tyrosine kinases (RTKs), includingVEGFR-2. The prevention or treatment of corneal disruptions or diseaseby a second active agent is achieved via moculation (e.g., activation)of EGFR.

Any of the disclosed diseases or conditions described herein can betreated or prevented by achieving target tissue concentration of fromabout 200 nM-about 2 μM of the disclosed compounds or pharmaceuticallyacceptable salts, formulation and/or suspension thereof. One embodimentof this application relates to a method for treating pathologicangiogenesis in the posterior segment of the eye, achieving targettissue concentration of about of about 200 nM-about 2 μM of thedisclosed compounds or pharmaceutically acceptable salts, and/orformulation thereof. Another iteration of this embodiment relates toachieving target tissue concentration of about 300 nM-about 2 μM of oneor more of the disclosed compounds or pharmaceutically acceptable salts,and/or formulation thereof.

In an embodiment of the present application about 0.2-about 1.0% (about2-about 10 mg/mL) of Compound-I formulated as a solution or suspension,upon administration, may effectively inhibit VEGFR-2 kinase function andprovide substantial blockade of a set of proangiogenic growth factorreceptors, including FGFRs1-3, Tie-2, and EphB-4. The 2-10 mg/mLconcentration of the Compound-I in the formulation provides effectivepharmacologically effective concentrations of drug to the centralchoroid and retina following 1-5 days of topical ocular delivery.

In some embodiments, the exposure time of Compound-I is between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments, the dosage regimen involves severalcourses of topical ocular administration of a formulation comprisingCompound-I to a subject for between 1 and 90 days or for longer than 90days (e.g., 4 months, 6 months, 8 months, or 12 months). For example,the dosage regimen involves once daily, twice daily, three times dailyor four times daily administration of the formulation for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). For example, the dosage regimen involves once, twice,three times, or four times administration of the formulation on everyother day (i.e., on day 1, 3, 5, 7 etc.) for up to 90 days. For example,the dosage regimen involves administering once on day 1, once or twiceon day 2-day 90. For example, the dosage regimen involves administeringonce, twice, three times, or four times on day 1, followed by once dailyfor 2-90 days. For example, the dosage regimen involves administeringonce, twice, three times, four times on day 1, followed by once, twice,three times, or four times on every other day (i.e., on day 1, 3, 5, 7etc.) for up to 90 days. For example, one dosage regimen involves onceper day or twice per day for 1, 2, 3, 4, or 5 consecutive days. Fortwice or three daily dosage regimen, subjects receive topical oculardose of a Compound-I formulation on days 1 and 4 approximately about 4,6, or 8 hours apart. In another embodiment, subjects receive topicalocular doses of a Compound-I formulation approximately about 4, 6, or 8hours apart for four consecutive days. In some embodiments, subjectsreceive one or two doses of topical ocular dose of Compound-Iformulation per day for 5 consecutive days. In yet other embodiments,subjects receive one or two doses of topical ocular dose of Compound-Iformulation for 5-90 consecutive days. In some embodiments, subjectsreceive one or two doses of topical ocular dose of Compound-Iformulation for at least 25 consecutive days. In one embodiment,subjects receive one or two topical ocular doses for at least 90consecutive days or more.

In some embodiments, the present application provides a formulation ofCompound-I or its free base and/or a second active agent administeredtopically to the anterior segment of the eye of the subject to treatAMD, pathologic CNV, and/or pathologic NV. For example, the formulationis administered to the eye of a subject 1, 2, 3, or 4 times daily. Inspecific embodiments, the formulation is administered to the eye of asubject 2 or 3 times daily. For example, the formulation is administeredto one eye or both eyes of a subject. For example, about 1 mg/ml of afirst active agent comprising formulation of the current disclosure isadministered twice a day (BID) to one eye or both eyes of a subject. Insome embodiments, about 1 mg/mL once a day (QD) or BID, about 2 mg/mL QDor BID, about 3 mg/mL QD or BID, about 4 mg/mL QD or BID, about 5 mg/mLQD or BID, about 6 mg/mL QD or BID, about 7 mg/mL QD or BID, about 8mg/mL QD or BID, about 9 mg/mL QD or BID, or about 10 mg/mL QD or BID ofis administered to one eye or both eyes of a subject.

For example, a formulation comprising about 1 mg/mL BID of Compound-Iand/or a second active agent is administered to one eye or both eyes ofa subject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 1 mg/mL QD of Compound-I and/or a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 1 mg/mL TID of Compound-I and/or a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 1mg/mL QID of Compound-I and/or a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 2 mg/mL BID ofCompound-I and/or a second active agent is administered to one eye orboth eyes of a subject for between 1 and 90 days or for longer than 90days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 2 mg/mL QD of Compound-Iand/or a second active agent is administered to one eye or both eyes ofa subject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 2 mg/mL TID of Compound-I and/or a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 2 mg/mL QID of Compound-I and/or a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 3mg/mL BID of Compound-I and/or a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 3 mg/mL QD ofCompound-I and/or a second active agent is administered to one eye orboth eyes of a subject for between 1 and 90 days or for longer than 90days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 3 mg/mL TID of Compound-Iand/or a second active agent is administered to one eye or both eyes ofa subject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 3 mg/mL QID of Compound-I and/or a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 4 mg/mL BID of Compound-I and/or a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 4mg/mL QD of Compound-I and/or a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 4 mg/mL TID ofCompound-I and/or a second active agent is administered to one eye orboth eyes of a subject for between 1 and 90 days or for longer than 90days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 4 mg/mL QID of Compound-Iand/or a second active agent is administered to one eye or both eyes ofa subject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 5 mg/mL BID of Compound-I and/or a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 5 mg/mL QD of Compound-I and/or a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 5mg/mL TID of Compound-I and/or a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 5 mg/mL QID ofCompound-I and/or a second active agent is administered to one eye orboth eyes of a subject for between 1 and 90 days or for longer than 90days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 6 mg/mL BID of Compound-Iand/or a second active agent is administered to one eye or both eyes ofa subject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 6 mg/mL QD of Compound-I and/or a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 6 mg/mL TID of Compound-I and/or a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 6mg/mL QID of Compound-I and/or a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 7 mg/mL BID of afirst active agent (e.g., Compound-I) and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments a formulation comprising about 7 mg/mLQD of a first active agent (e.g., Compound-I) and a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 7mg/mL TID of a first active agent (e.g., Compound-I) and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments a formulation comprisingabout 7 mg/mL QID of a first active agent (e.g., Compound-I) and asecond active agent is administered to one eye or both eyes of a subjectfor between 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 8 mg/mL BID of a first active agent (e.g., Compound-I)and a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments aformulation comprising about 8 mg/mL QD of a first active agent (e.g.,Compound-I) and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodimentsa formulation comprising about 8 mg/mL TID of a first active agent(e.g., Compound-I) and a second active agent is administered to one eyeor both eyes of a subject for between 1 and 90 days or for longer than90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments a formulation comprising about 8 mg/mL QID of a first activeagent (e.g., Compound-I) and a second active agent is administered toone eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments a formulation comprising about 9 mg/mL BID of afirst active agent (e.g., Compound-I) and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments a formulation comprising about 9 mg/mLQD of a first active agent (e.g., Compound-I) and a second active agentis administered to one eye or both eyes of a subject for between 1 and90 days or for longer than 90 days (e.g., 4 months, 6 months, 8 months,or 12 months). In some embodiments a formulation comprising about 9mg/mL TID of a first active agent (e.g., Compound-I) and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments a formulation comprisingabout 9 mg/mL QID of a first active agent (e.g., Compound-I) and asecond active agent is administered to one eye or both eyes of a subjectfor between 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments a formulationcomprising about 10 mg/mL QD of a first active agent (e.g., Compound-I)and a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). The dosage regimen forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months) may be any of the regimens involvingconsecutive or alternate days described in the paragraph above. In someembodiments, the formulation of the present application is administeredQD, BID, TID, or QID when administered at low doses (e.g., 1 mg/mL, 2mg/mL, 3 mg/mL, 4 mg/mL, or 5 mg/mL), and QD or BID at high doses (e.g.,6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, or 10 mg/mL).

In other embodiment, a 1 mg/mL BID of Compound-I formulation and asecond active agent is administered to one eye or both eyes of a subjectfor between 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments, a 1 mg/mL QD ofCompound-I formulation and a second active agent is administered to oneeye or both eyes of a subject for between 1 and 90 days or for longerthan 90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments, a 2 mg/mL BID of Compound-I formulation and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments, a 2 mg/mL QD of Compound-Iformulation and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodiments,a 3 mg/mL BID of Compound-I formulation and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments, a 3 mg/mL QD of Compound-I formulationand a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments a 4 mg/mLBID of Compound-I formulation and a second active agent is administeredto one eye or both eyes of a subject for between 1 and 90 days or forlonger than 90 days (e.g., 4 months, 6 months, 8 months, or 12 months).In some embodiments, a 4 mg/mL QD of Compound-I formulation and a secondactive agent is administered to one eye or both eyes of a subject forbetween 1 and 90 days or for longer than 90 days (e.g., 4 months, 6months, 8 months, or 12 months). In some embodiments, a 5 mg/mL BID ofCompound-I formulation and a second active agent is administered to oneeye or both eyes of a subject for between 1 and 90 days or for longerthan 90 days (e.g., 4 months, 6 months, 8 months, or 12 months). In someembodiments, a 5 mg/mL QD of Compound-I formulation and a second activeagent is administered to one eye or both eyes of a subject for between 1and 90 days or for longer than 90 days (e.g., 4 months, 6 months, 8months, or 12 months). In some embodiments, a 6 mg/mL BID of Compound-Iformulation and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodiments,a 6 mg/mL QD of Compound-I formulation and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments, a 7 mg/mL BID of Compound-I formulationand a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments, a 7mg/mL QD of Compound-I formulation and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments, a 8 mg/mL BID of Compound-I formulationand a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments, a 8mg/mL QD of Compound-I formulation and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments, a 9 mg/mL BID of Compound-I formulationand a second active agent is administered to one eye or both eyes of asubject for between 1 and 90 days or for longer than 90 days (e.g., 4months, 6 months, 8 months, or 12 months). In some embodiments, a 9mg/mL QD of Compound-I formulation and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). In some embodiments, a 10 mg/mL BID of Compound-Iformulation and a second active agent is administered to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodiments,a 10 mg/mL QD of Compound-I formulation and a second active agent isadministered to one eye or both eyes of a subject for between 1 and 90days or for longer than 90 days (e.g., 4 months, 6 months, 8 months, or12 months). The dosage regimen for between 1 and 90 days or for longerthan 90 days (e.g., 4 months, 6 months, 8 months, or 12 months) may beany of the regimens involving consecutive or alternate days described inthe paragraph above.

The present application provides formulations as shown in Table 13 foradministering to one eye or both eyes of a subject.

TABLE 13 10 mM Sodium Dose/ Formula Compound-I Formula II/ KLEPTOSE ®Phosphate Chloride Day II (%) (%) Compound-I:CD HPB (%) (%) (%)* pH QD0.40 0.427 1:8 8.411 0.142 QS to about 6 285 mOsm QD 0.60 0.641 1:812.626 0.142 QS to about 6 285 mOsm BID 0.10 0.107 1:8 2.103 0.142 QS toabout 6 285 mOsm BID 0.20 0.214 1:8 4.205 0.142 QS to about 6 285 mOsmBID 0.30 0.321 1:8 6.308 0.142 QS to about 6 285 mOsm BID 0.40 0.427 1:88.411 0.142 QS to about 6 285 mOsm *QS = quantity sufficient forachieving the osmolality

In some embodiments, the formulation of Formula II or Compound-I isadministered to one eye or both eyes of a subject. For example, about0.2%-about 1.0% (w/v) of the compound of Formula II or about 0.1%-1.2%(w/v) of Compound-I comprising formulation of the current disclosure isadministered once a day (QD) or twice a day (BID) to one eye or botheyes of a subject for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months). In some embodiments,Formula II compound or Compound-I is complexed with a complexing agent,e.g., cyclodextrin (e.g., KLEPTOSE® HPB (%)) in ratio of about 1:8, inwhich about 2%-13% (w/v) cyclodextrin (e.g., KLEPTOSE® HPB (%)) is addedto the formulation. The formulation further comprises about 0.1%-about0.2% buffer, e.g., 10 mM phosphate buffer. The desired osmolality of theformulation is about 200-about 300 mOsm, achieved by adding quantitysufficient to achieve the osmolality with a salt, e.g., sodium chloride.The pH of the formulation is about 6.0 at or under about 40° C. Thedosage regimen for between 1 and 90 days or for longer than 90 days(e.g., 4 months, 6 months, 8 months, or 12 months) may be any of theregimens involving consecutive or alternate days described in theparagraph above.

In some embodiments, the Formula II compound or Compound-I formulationfurther comprises a second active agent. In some embodiment, Formula IIcompound or Compound-I formulation is administered with a second activeagent. The second active agent, e.g., nicotinic acid, nicotinamide, orvitamin K or a combination thereof, at a specific dose modulates (e.g.,activates) directly or indirectly, ErbB receptor tyrosine kinase (RTK)including, EGFR, HER2, HER3, and HER4. The modulation (e.g., activation)of one or more ErbB RTKs by the formulation of the present application,at a specific dose, simultaneous and has a synergistic effect, and iseffective in the prevention or treatment of corneal disruptions ordiseases, (e.g., corneal ulcers, corneal epithelial defects, keratitis,etc.) in the anterior segment of the eye.

The methods of the present application are combined with the standard ofcare, including but not limited to laser treatment and treatment withinjectable anti-neovascular agents.

Particle Compositions and Formulations Comprising the ParticleCompositions

The present application relates to a pharmaceutical compositioncomprising particles of an active agent of the present application(e.g., a first active agent (e.g., Formula II or Compound-I) and/or asecond active agent), or a pharmaceutically acceptable salt thereof,wherein the particles have a mean diameter of between 100 nm and 100 μm.In some embodiments, the particles have a mean diameter of between 20 μmand 90 μm. In some embodiments, the particles have a mean diameter ofbetween 20 μm and 80 μm. In some embodiments, the particles have a meandiameter of between 20 μm and 70 μm. In some embodiments, the particleshave a mean diameter of between 30 μm and 70 μm. In some embodiments,the particles have a mean diameter of between 30 μm and 60 μm. In someembodiments, the particles have a mean diameter of between 30 μm and 50μm. In some embodiments, the particles have a mean diameter of between30 μm and 40 μm. In some embodiments, the particles have a mean diameterof between 50 μm and 70 μm. In some embodiments, the particles have amean diameter of between 50 μm and 60 μm. In some embodiments, theparticles have a mean diameter of at least 30 μm. In some embodiments,the particles have a mean diameter of about 30 μm, about 35 μm, about 40μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, orabout 70 μm. In some embodiments, the particles have a mean diameter ofabout 30 μm, about 35 μm, about 50 μm, or about 60 μm. In someembodiments, the particles have a mean diameter of between 100 nm and 8μm. In some embodiments, the particles have a mean diameter of between100 nm and 200 nm. In some embodiments, the particles have a meandiameter of at most 150 nm. In some embodiments, the particles have amean diameter of about 150 nm, about 140 nm, about 130 nm, about 120 nm,about 110 nm, or about 100 nm. In other embodiments, the particles havea mean diameter of between 1 μm and 5 μm. In some embodiments, theparticles have a mean diameter of between 2 μm and 4 μm. In someembodiments, the particles have a mean diameter of about 1 μm, about 2μm, about 3 μm, about 4 μm, or about 5 μm. In some embodiments, theparticles have a mean diameter of about 3 μm.

In some embodiments, at least 90% of the particles have a diameter of 70μm or less. In some embodiments, at least 90% of the particles have adiameter of 60 μm or less. In some embodiments, at least 90% of theparticles have a diameter of 10 μm or less. In some embodiments, atleast 90% of the particles have a diameter of 9 μm or less. In someembodiments, at least 90% of the particles have a diameter of 8 μm orless. In some embodiments, at least 90% of the particles have a diameterof 7 μm or less. In some embodiments, at least 90% of the particles havea diameter of 6 μm or less. In some embodiments, at least 90% of theparticles have a diameter of 5 μm or less. In some embodiments, at least90% of the particles have a diameter of 4 μm or less. In someembodiments, at least 90% of the particles have a diameter of 300 nm orless. In some embodiments, at least 90% of the particles have a diameterof 200 nm or less.

In some embodiments, the pharmaceutical composition comprises particlesof a first active agent (e.g., a first active agent (e.g., Formula II orCompound-I)) and particles of a second active agent (e.g., nicotinicacid, nicotinamide, vitamin K, or a combination thereof). In someembodiments, the pharmaceutical composition comprises particles of afirst active agent (e.g., a first active agent (e.g., Formula II orCompound-I)) and particles of vitamin K (e.g., menadione).

In some embodiments, a first active agent (e.g., Formula II orCompound-I) and a second active agent (e.g., nicotinic acid,nicotinamide, vitamin K, or a combination thereof) are roller milledtogether to form particles comprising the first active agent and thesecond active agent. In some embodiments, the first active agent and thesecond active agent are roller milled separately, and the particlescomprising the first active agent and the particles comprising thesecond active agent are then mixed or roller milled together. In someembodiments, the first active agent or the second active agent is rollermilled first, and the particles are then added to the other active agentfor further roller milling.

In some embodiments, the pharmaceutical composition comprising particlesof an active agent of the present application (e.g., a first activeagent or a second active agent) further comprises one or moreexcipients. The excipient can be selected from any suitable excipientknown in the art, for example, for preparing an ophthalmic formulation.In some embodiments, the excipient is selected from Polysorbate (Tween)80, Poloxamer (Pluronic) F-127, Hypromellose (HydroxypropylMethylcellulose or HPMC), Povidone (PVP K-29/32 or K-30), and Tyloxapol,and a combination thereof. In some embodiments, the excipient isselected from HPMC, Tween 80, Pluronic F-127, and Tyloxapol, and acombination thereof.

In some embodiments, the particles comprising a first active agent(e.g., Formula II or Compound-I) and the particles comprising a secondactive agent (e.g., nicotinic acid, nicotinamide, vitamin K, or acombination thereof) comprise the same excipient(s).

In some embodiments, the pharmaceutical composition comprising particlesof an active agent of the present application (e.g., a first activeagent or a second active agent) further comprises a surfactant, such asbenzalkonium chloride (BAC).

In some embodiments, the pharmaceutical composition comprising particlesof an active agent of the present application (e.g., a first activeagent or a second active agent) further comprises an excipient (e.g.,for enhancing bioavailability of the active agent). In some embodiments,the excipient is hydroxyethyl cellulose (HEC). In some embodiments, theHEC is present in an amount of about 0.1%-about 1%, about 0.1%-about0.9%, about 0.1%-about 0.8%, about 0.1%-about 0.7%, about 0.1%-about0.6%, about 0.1%-about 0.5%, about 0.1%-about 0.4%, or about 0.1%-about0.3%. In some embodiments, the HEC is present in an amount of about 0.2%or about 0.3%.

In some embodiments, the particles of the present application compriseFormula II (i.e., free base of Compound-I). In other embodiments, theparticles of the present application comprises Compound-I.

In some embodiments, the particles of the present application areprepared by roller milling. Factors that may affect the size of theparticles include, but are not limited to, the use of the free base or asalt of the active agent (e.g., Compound-I vs. Formula II), the additionof an excipient, the speed of the roller during milling, the size of themilling media, and duration of the milling.

In some embodiments, the particles of the present application areprepared without roller milling.

In some embodiments, the particles of an active agent of the presentapplication (e.g., a first active agent (e.g., Formula II or Compound-I)and/or a second active agent) are sterilized. In some embodiments, thesterilization is conducted with gamma irradiation. In some embodiments,the particles of an active agent of the present application (e.g., afirst active agent (e.g., Formula II or Compound-I) and/or a secondactive agent) are stable after the sterilization (e.g., remainingsubstantially pure of any impurities or degradation products as a resultof the sterilization).

The present application relates to a suspension formulation comprising apharmaceutical composition, wherein the pharmaceutical compositioncomprises particles of an active agent of the present application (e.g.,a first active agent (e.g., Formula II or Compound-I) and/or a secondactive agent), or a pharmaceutically acceptable salt thereof, asdescribed herein.

In some embodiments, the suspension formulation comprises particles of afirst active agent (e.g., Formula II or Compound-I), wherein the firstactive agent is at a concentration of about 0.1 mg/mL-about 10 mg/mL. Insome embodiments, the first active agent is at a concentration of about0.2 mg/mL-about 10 mg/mL, about 0.5 mg/mL-about 10 mg/mL, about 1mg/mL-about 10 mg/mL, about 2 mg/mL-about 9 mg/mL, about 2 mg/mL-about 8mg/mL, about 3 mg/mL-about 8 mg/mL, about 3 mg/mL-about 7 mg/mL, about 3mg/mL-about 6 mg/mL, about 4 mg/mL-about 6 mg/mL, or about 4 mg/mL-about5 mg/mL of a first active agent (e.g., Formula II or Compound-I). Insome embodiments, the first active agent is at a concentration of about0.1 mg/mL, 0.3 mg/mL, 0.5 mg/mL, about 1 mg/mL, about 2 mg/mL, about 3mg/mL, about 4 mg/mL, about 6 mg/mL, or about 10 mg/mL. In someembodiments, the first active agent is at a concentration of about 1mg/mL-about 4 mg/mL or about 2 mg/mL-about 4 mg/mL. In some embodiments,the first active agent is at a concentration of about 2 mg/mL or about 4mg/mL.

In some embodiments, the suspension formulation comprising particles ofa first active agent (e.g., Formula II or Compound-I) further comprisesa second active agent (e.g., nicotinic acid, nicotinamide, vitamin K, ora combination thereof). In some embodiments, the second active agent isvitamin K (e.g., menadione). In some embodiments, the second activeagent is present in an amount of less than 10 μM. In some embodiments,the second active agent is present in an amount of about 0.5 μM, about0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 2μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8μM, or about 9 μM. In some embodiments, the second active agent ispresent in an amount of about 1 μM.

In some embodiments, the suspension formulation comprising particles ofa first active agent (e.g., Formula II or Compound-I) further comprisesan excipient selected from Polysorbate (Tween) 80, Poloxamer (Pluronic)F-127, Hypromellose (Hydroxypropyl Methylcellulose or HPMC), Povidone(PVP K-29/32 or K-30), and Tyloxapol, and a combination thereof. In someembodiments, the excipient is Pluronic F-127, Tween 80, HPMC, orTyloxapol, or a combination thereof. In some embodiments, the excipientis present in a concentration of about 0.01%-about 0.2%, about0.01%-about 0.15%, about 0.01%-about 0.12%, about 0.01%-about 0.1%,about 0.01%-about 0.09%, about 0.02%-about 0.09%, about 0.03%-about0.09%, about 0.04%-about 0.09%, or about 0.04%-about 0.08%. In someembodiments, the suspension formulation comprises about 0.08% or about0.04% Pluronic F-127. In some embodiments, the suspension formulationcomprises about 0.08% HPMC. In other embodiments, the suspensionformulation comprises about 0.04% Tyloxapol.

In some embodiments, the suspension formulation comprising particles ofa first active agent (e.g., Formula II or Compound-I) further comprisesa buffering agent. In some embodiments, the buffering agent is selectedfrom phosphate buffers, borate buffers, citrate buffers, tartratebuffers, acetate buffers, amino acids, sodium acetate, sodium citrate,Tris buffers, and the like. In some embodiments, the buffering agent isTris. In some embodiments, the buffering agent is present in aconcentration of about 0.1%-about 2%, about 0.2%-about 1.8%, about0.3%-about 1.6%, about 0.4%-about 1.4%, about 0.4%-about 1.2%, about0.4%-about 1%, about 0.4%-about 0.8%, about 0.4%-about 0.7%, or about0.5%-about 0.7%. In some embodiments, the suspension formulationcomprises about 0.6% Tris.

In some embodiments, the suspension formulation comprising particles ofa first active agent (e.g., Formula II or Compound-I) further comprisesan osmolality adjusting reagent. In some embodiments, the osmolalityadjusting reagent is glycerol. In some embodiments, the osmolalityadjusting reagent is present in an amount of about 1%-about 10%, about2%-about 10%, about 3%-about 10%, about 4%-about 10%, about 5%-about10%, about 2%-about 9%, about 2%-about 8%, about 2%-about 7%, about2%-about 6%, about 2%-about 5%, about 2%-about 4%, or about 2%-about 3%.In some embodiments, the suspension formulation comprises about 2% orabout 2.5% glycerol.

In some embodiments, the suspension formulation comprising particles ofa first active agent (e.g., Formula II or Compound-I) has a pH of lessthan 7.5. In some embodiments the pH is between about 6.0-about 7.0. Insome embodiments the pH is about 6.0 or about 7.0.

In some embodiments, the suspension formulation comprising particles ofa first active agent (e.g., Formula II or Compound-I) further compriseshydroxyethyl cellulose (HEC). In some embodiments, the HEC is present inan amount of about 0.1%-about 1%, about 0.1%-about 0.9%, about0.1%-about 0.8%, about 0.1%-about 0.7%, about 0.1%-about 0.6%, about0.1%-about 0.5%, about 0.1%-about 0.4%, or about 0.1%-about 0.3%. Insome embodiments, the HEC is present in an amount of about 0.2% or about0.3%.

One of a first active agent, a second active agent, and one or moreexcipients described in the present application can be present at anyconcentration or level described herein in combination with theremainder of the first active agent, the second active agent, and one ormore excipients described in the present application at anyconcentration or level described herein.

In some embodiments, the formulation of the present applicationcomprises a first active agent at about 0.1% to about 1.2% (or any rangein between as described herein), Tris at about 0.4%-about 0.8% (or anyrange in between as described herein), glycerol at about 2%-about 4% (orany range in between as described herein), HEC at about 0.1%-about 0.3%(or any range in between as described herein), and HPMC at about0.04%-about 0.09% (or any range in between as described herein). In someembodiments, the first active agent is Formula II or Compound-I. In someembodiments, the formulation of the present application comprisesFormula II or Compound-I at about 0.4%, Tris at about 0.6%, glycerol atabout 2%, HEC at about 0.2%, and HPMC at about 0.08%. In someembodiments, the formulation comprises particles of the first activeagent wherein the particles have a mean diameter of between 30 μm and 60μm. In some embodiments, the formulation comprises particles of thefirst active agent wherein the particles have a mean diameter of between50 m and 60 μm. In some embodiments, the formulation comprises particlesof the first active agent wherein the particles have a mean diameter ofabout 50 μm or 60 μm. In some embodiments, the formulation has a pH ofless than 7.0. In some embodiments, the formulation has a pH of about 6.

In some embodiments, the formulation of the present applicationcomprises a first active agent at about 0.1% to about 1.2% (or any rangein between as described herein), a second active agent at about0.00001%-about 0.0001% (or any range in between as described herein),Tris at about 0.4%-about 0.8% (or any range in between as describedherein), glycerol at about 2%-about 4% (or any range in between asdescribed herein), HEC at about 0.1%-about 0.3% (or any range in betweenas described herein), and HPMC at about 0.04%-about 0.09% (or any rangein between as described herein). In some embodiments, the first activeagent is Formula II or Compound-I. In some embodiments, the secondactive agent is vitamin K₃ (e.g., menadione). In some embodiments, theformulation of the present application comprises Formula II orCompound-I at about 0.4%, vitamin K₃ at about 0.000086%, Tris at about0.6%, glycerol at about 2%, HEC at about 0.2%, and HPMC at about 0.08%.In some embodiments, the formulation comprises particles of the firstactive agent wherein the particles have a mean diameter of between 30 μmand 60 μm. In some embodiments, the formulation comprises particles ofthe first active agent wherein the particles have a mean diameter ofbetween 50 μm and 60 μm. In some embodiments, the formulation comprisesparticles of the first active agent wherein the particles have a meandiameter of about 50 μm or 60 μm. In some embodiments, the particle alsocomprises the second active agent. In some embodiments, the formulationhas a pH of less than 7.0. In some embodiments, the formulation has a pHof about 6.

Indications and Methods of Treatment

Disclosed are methods for the treatment of diseases or conditions of theeye. The disclosed methods relate to treating, preventing, orcontrolling ocular neovascularization (NV), or treating a disease orcondition that is related to the onset of NV by administering to asubject one or more of the disclosed compounds (e.g., a first activeagent (e.g., a compound of Formula I or II), and optionally a secondactive agent), and formulations thereof.

One aspect of the disclosed method relates to treating or preventing NVby administering to a subject an effective amount of one or more of thedisclosed compounds of Formula I or II, or pharmaceutically acceptablesalts, and optionally a second active agent (e.g., vitamin K, nicotinicacid, or nicotinamide, or a combination thereof), and/or formulationsthereof. One embodiment of this aspect relates to a method for treatingNV by administering to a subject a composition of: a) an effectiveamount of one or more of the disclosed compounds of Formula I or II, orpharmaceutically acceptable salts, and optionally a second active agent(e.g., vitamin K, nicotinic acid, or nicotinamide, or a combinationthereof), and/or formulations thereof, and optionally b) one or morecarriers or compatible excipients.

The disclosed methods relate to preventing or controlling pathologicocular neovascularization (NV), or treating a disease or condition thatis related to the onset of NV by administering to a subject one or moreof the disclosed compounds of Formula I or II, and optionally a secondactive agent (e.g., vitamin K, nicotinic acid, or nicotinamide, or acombination thereof), and formulations thereof.

The current embodiments provide use of a formulation of Compound-I orits free base (Formula II), and optionally a second active agent (e.g.,vitamin K, nicotinic acid, or nicotinamide, or a combination thereof),for the manufacture of a medicament for treating a subject with aposterior segment disease vasculopathic or inflammatory disease of theeye. These include for example, diabetic retinopathy (includingbackground diabetic retinopathy, proliferative diabetic retinopathy anddiabetic macular edema); age-related macular degeneration (AMD)(including neovascular (wet/exudative) AMD, dry AMD, and GeographicAtrophy); pathologic choroidal neovascularization (CNV) from anymechanism (i.e. high myopia, trauma, sickle cell disease; ocularhistoplasmosis, angioid streaks, traumatic choroidal rupture, drusen ofthe optic nerve, and some retinal dystrophies); pathologic retinalneovascularization from any mechanism (i.e., sickle cell retinopathy,Eales disease, ocular ischemic syndrome, carotid cavernous fistula,familial exudative vitreoretinopathy, hyperviscosity syndrome,idiopathic occlusive arteriolitis; birdshot retinochoroidopathy, retinalvasculitis, sarcoidosis, or toxoplasmosis); uveitis; retinal veinocclusion (central or branch); ocular trauma; surgery induced edema;surgery induced neovascularization; cystoid macular edema; ocularischemia; retinopathy of prematurity; Coat's disease; sickle cellretinopathy and/or neovascular glaucoma. In one embodiment, the diseaseof the eye is AMD. In one embodiment, the diseases of the eye arise fromor are exacerbated by ocular angiogenesis and/or neovascularization.

In one aspect of the present application the formulation is used in thetreatment of age-related macular degeneration (AMD) (includingneovascular (wet/exudative) AMD, dry AMD, and Geographic Atrophy). Thesolutions or suspensions are used in the treatment of neovascular(exudative or wet) AMD. In another embodiment, the solutions orsuspensions are used to treat dry AMD. In yet another embodiment, thesolutions or suspensions are used to treat Geographic Atrophy.

The formulation of the present application prevents, delays, or treatsthe onset of pathologic choroidal neovascularization (CNV) from anymechanism (i.e. high myopia, trauma, sickle cell disease; ocularhistoplasmosis, angioid streaks, traumatic choroidal rupture, drusen ofthe optic nerve, and some retinal dystrophies) in subjects.

The formulation of the present application delays onset, preventsprogression, or treats formation of pathological choroidalneovascularization (CNV) below the neurosensory retina. The formulationof the present application is effective in treating CNV.

One aspect of this method relates to treating or preventing ocularneovascularization by administering to a subject an effective amount ofone or more of the disclosed compounds of Formula I or II, orpharmaceutically acceptable salts thereof, and optionally a secondactive agent (e.g., vitamin K, nicotinic acid, or nicotinamide, or acombination thereof). One embodiment of this aspect relates to a methodfor treating ocular edema and neovascularization by administering to asubject a composition of: a) an effective amount of one or more of thedisclosed compounds of Formula I or II, or pharmaceutically acceptablesalts, and optionally a second active agent (e.g., vitamin K, nicotinicacid, or nicotinamide, or a combination thereof), and/or formulationsthereof, and optionally b) one or more carriers or compatibleexcipients.

The disclosed methods also relate to preventing or controlling ocularedema or treating a disease or condition that is related to the onset ofocular edema by administering to a subject one or more or the disclosedcompounds of Formula I or II and optionally a second active agent (e.g.,vitamin K, nicotinic acid, or nicotinamide, or a combination thereof).

One aspect of this method relates to treating or preventing ocular edemaby administering to a subject an effective amount of one or more of thedisclosed compounds of Formula I or II, or pharmaceutically acceptablesalts thereof, and optionally a second active agent (e.g., vitamin K,nicotinic acid, or nicotinamide, or a combination thereof). Oneembodiment of this aspect relates to a method for treating ocular edemaby administering to a subject a composition of: a) an effective amountof one or more of the disclosed compounds of Formula I or II, orpharmaceutically acceptable salts, and optionally a second active agent(e.g., vitamin K, nicotinic acid, or nicotinamide, or a combinationthereof), and/or formulations thereof, and optionally b) one or morecarriers or compatible excipients.

Another disclosed method relates to preventing or controlling retinaledema or retinal neovascularization or treating a disease or conditionthat is related to the onset of retinal edema or retinalneovascularization by administering to a subject one or more or thedisclosed compounds of Formula I or II and optionally a second activeagent (e.g., vitamin K, nicotinic acid, or nicotinamide, or acombination thereof). One aspect of this method relates to treating orpreventing retinal edema or retinal neovascularization by administeringto a subject an effective amount of one or more of the disclosedcompounds of Formula I or II, or pharmaceutically acceptable saltsthereof, and optionally a second active agent (e.g., vitamin K,nicotinic acid, or nicotinamide, or a combination thereof). Oneembodiment of this aspect relates to a method for treating retinal edemaor retinal neovascularization by administering to a subject acomposition of: a) an effective amount of one or more of the disclosedcompounds of Formula I or II, or pharmaceutically acceptable salts, andoptionally a second active agent (e.g., vitamin K, nicotinic acid, ornicotinamide, or a combination thereof), and/or formulations thereof,and optionally b) one or more carriers or compatible excipients.

Another embodiment of this aspect relates to a method for delaying orpreventing progression of non-proliferative retinopathy to proliferativeretinopathy by administering to a subject a composition of: a) aneffective amount of one or more of the disclosed compounds of Formula Ior II, or pharmaceutically acceptable salts, and optionally a secondactive agent (e.g., vitamin K, nicotinic acid, or nicotinamide, or acombination thereof), and/or formulations thereof, and optionally b) oneor more carriers or compatible excipients.

One aspect of the disclosed methods relates to diseases that are adirect or indirect result of diabetes, inter alia, diabetic macularedema and diabetic retinopathy. The ocular vasculature of the diabeticbecomes unstable over time leading to conditions such asnon-proliferative retinopathy, macular edema, and proliferativeretinopathy. As fluid leaks into the center of the macula, the part ofthe eye where sharp, straight-ahead vision occurs, the buildup of fluidand the associated protein begin to deposit on or under the macula. Thisresults in swelling that causes the subject's central vision togradually become distorted. This condition is referred to as “macularedema.” Another condition that may occur is non-proliferativeretinopathy in which vascular changes, such as microaneurysms, outsidethe macular region of the eye may be observed. During proliferative DR,pathologic new blood vessels grow in and up from the retina into to thevitreous body, where these abnormal vessels may alter retinal morphologyin the macula, and/or hemorrhage into the vitreous and obscure thevisual axis.

A further disclosed method relates to treating, preventing orcontrolling diabetic retinopathy or treating a disease or condition thatis related to the onset of diabetic retinopathy by administering to asubject one or more or the disclosed compounds of Formula I or II, andoptionally a second active agent (e.g., vitamin K, nicotinic acid, ornicotinamide, or a combination thereof).

One aspect of the disclosed method relates to treating or preventingdiabetic retinopathy by administering to a subject an effective amountof one or more of the disclosed compounds of Formula I or II, orpharmaceutically acceptable salts thereof, and optionally a secondactive agent (e.g., vitamin K, nicotinic acid, or nicotinamide, or acombination thereof). One embodiment of this aspect relates to a methodfor treating diabetic retinopathy by administering to a subject acomposition of: a) an effective amount of one or more of the disclosedcompounds of Formula I or II, or pharmaceutically acceptable salts, andoptionally a second active agent (e.g., vitamin K, nicotinic acid, ornicotinamide, or a combination thereof), and/or formulations thereof,and optionally b) one or more carriers or compatible excipients.

Diabetic proliferative retinopathy is characterized byneovascularization. The new blood vessels are fragile and aresusceptible to bleeding. The result is scarring of the retina, as wellas occlusion or total blockage of the light pathway through the eye dueto the abnormal formation of new blood vessels. Typically subjectshaving diabetic macular edema are suffering from the non-proliferativestage of diabetic retinopathy; however, it is not uncommon for subjectsto only begin manifesting macular edema at the onset of theproliferative stage.

Yet a further disclosed method relates to preventing or controllingdiabetic macular edema or treating a disease or condition that isrelated to the onset of diabetic macular edema by administering to asubject one or more or the disclosed compounds of Formula I or II andoptionally a second active agent (e.g., vitamin K, nicotinic acid, ornicotinamide, or a combination thereof).

One aspect of this method relates to treating or preventing diabeticmacular edema by administering to a subject an effective amount of oneor more of the disclosed compounds of Formula I or II, orpharmaceutically acceptable salts, and optionally a second active agent(e.g., vitamin K, nicotinic acid, or nicotinamide, or a combinationthereof), or formulations thereof. One embodiment of this aspect relatesto a method for treating diabetic macular edema by administering to asubject a composition of: a) an effective amount of one or more of thedisclosed compounds of Formula I or II, or pharmaceutically acceptablesalts, and optionally a second active agent (e.g., vitamin K, nicotinicacid, or nicotinamide, or a combination thereof), and/or formulationsthereof, and b) one or more carriers or compatible excipients.

Another aspect of the disclosed method relates to treating or preventingNV and treating and/or preventing corneal disruptions or diseases byadministering to a subject an effective amount of one or more of thedisclosed compounds of Formula I or II, or pharmaceutically acceptablesalts, a second active agent, and/or formulations thereof. Oneembodiment of this aspect relates to a method for treating NV andtreating and/or preventing corneal disruptions or diseases byadministering to a subject a composition of: a) an effective amount ofone or more of the disclosed compounds of Formula I or II, orpharmaceutically acceptable salts, and/or formulations thereof, b) asecond active agent, and optionally c) one or more carriers orcompatible excipients. In one embodiment, the corneal disruption ordisease is caused by a compound of Formula I or II.

Kits

Also disclosed are kits of the disclosed compounds and compositions fordrug delivery into a human, mammal, or cell. The kits can comprise oneor more packaged unit doses of a composition comprising one or morecompounds of Formula I or II and one or more packaged unit doses of asecond active agent to be delivered into a human, mammal, or cell. Theunit dosage ampoules or multi-dose containers, in which the compounds ofFormula I or II or the second active agent to be delivered are packagedprior to use, can comprise a hermetically sealed container enclosing anamount of the active agent or pharmaceutically acceptable salt, orformulation thereof, suitable for a pharmaceutically effective dosethereof, or multiples of an effective dose. The compounds can bepackaged as a sterile formulation, and the hermetically sealed containeris designed to preserve sterility of the formulation until use.

The kit of the present application has a single-use eye drop dispenserbottle for delivery of ophthalmic formulation. In an alternativeembodiment, the kit of the present application has a multi-use eye-dropdispenser bottle. The multi-dose dispenser bottle has appropriate amountof anti-infective and/or preservative agent, for example without beinglimited to, 0.005% BAK. The ophthalmic dispenser of the presentapplication has a top and a cap. The container of the presentapplication has a semi-transparent LDPE ophthalmic dispenser bottle witha LDPE dropper tip and HDPE cap. The container may be of other type andform as needed and/or as used in the art.

The following examples are illustrative, but not limiting, of themethods and compositions of the present application. Other suitablemodifications and adaptations of the variety of conditions andparameters normally encountered in therapy and that are obvious to thoseskilled in the art are within the spirit and scope of the embodiments.

General Methodology

Roller Mill

The horizontal roller mill consists of multiple motor driven rollerscontained within a metal housing. Individual containers placed betweenthe rollers will rotate at an rpm determined by the speed of the rollersand the diameter of the container. Drug slurry consisting of API, andoptionally stabilizers, water, and/or milling media are added to thecontainer before being placed between the rollers. The media used arebeads of various sizes, e.g., from 800 microns to 3000 microns, and canbe made with Yttria Zirconium. After milling, the dispersion isseparated from the media by transferring the contents to a centrifugetube insert fitted with a screen mesh. After centrifugation, e.g., atapproximately 300×G for approximately 5 minutes, the dispersion iscollected below the mesh (which retained the media).

Optical Microscopy (OM)

The morphology and size distribution of the particle compositions can beassessed by optical microscopy, and photomicrographs of particles can betaken, for example, using an Olympus BX51 system equipped with an oilimmersion 100× objective (1000× magnification). A calibration bar (from1 um to 100 um) can be set as a comparator on each photomicrograph.

Particle Size Distribution (PSD)

Particle size distribution can be analyzed using laser diffraction lightscattering, e.g., with a Horiba LA-950 V2. Generic assumptions are madein setting conditions and the refractive index value. The distributionsare volume based. Sample density is adjusted to a generic range ofpercent transmission on the blue LED light source. A small sample cell(filled with water) can be used rather than the flow through cell, tominimize sample quantity.

Calculation

The following equation was used to determine the volume of diluent(50:50 methanol:water) required in order to prepare choroid, retina, andcornea samples at a specified tissue concentration.Vol._(Diluent)=(Mass_(Tissue)/Conc._(Tissue))−Vol._(Tissue)  Equation 1Where: Conc._(Tissue)=Desired tissue concentration (mg/mL)

-   -   Mass_(Tissue)=Mass of tissue (mg)    -   Vol._(Diluent)=Volume of diluent (50:50 methanol:water) (mL)    -   Vol._(Tissue)=Volume of tissue (mL), assuming density of 1.0        g/mL

To calculate the concentration in ng/g (ng of drug/g of tissue), thefollowing equation was used:Conc._(ng/g)=Conc._(ng/mL)×(Vol._(Total)/Mass_(Tissue))  Equation 2Where: Conc._(ng)/g=Calculated concentration (ng of drug/g of tissue)

-   -   Conc._(ng/mL)=Calculated concentration (ng of drug/mL of        homogenate)    -   Vol._(Total)=Total volume of tissue homogenate (mL)    -   Mass_(Tissue)=Mass of tissue (g)

To then calculate concentration of drug in tissue* in μM (μmol ofdrug/volume of tissue), the following equation was used, assuming atissue density of 1 g/mL: *Tissue=choroid, retina, or corneaConc._(μM)=Conc._(ng/g)/MW  Equation 3Where: Conc._(μM)=Calculated concentration (μmol of drug/volume oftissue)

-   -   Conc._(ng/g)=Calculated concentration (ng of drug/g of tissue)    -   MW=Molecular weight (g/mole)

To calculate concentration of drug in fluid** in M (μmol of drug/volumeof fluid), the following equation was used: **Fluid=plasmaConc._(μM)=Conc._(ng/mL)/MW  Equation 4Where: Conc._(μM)=Calculated concentration (μmol of drug/volume offluid)

-   -   Conc._(ng/mL)=Calculated concentration (ng of drug/mL of fluid)    -   MW=Molecular weight (g/mole)

Note: any sample concentrations <LLOQ within the body of this reportwere dropped or excluded for the calculation of the statistical valuesreported (average, standard deviation, and percent coefficient ofvariance).

Draize Eye Irritation Scoring System for Cornea, Iris, and Conjunctiva

Cornea A. Opacity - Degree of density (area which is most dense is takenfor reading) Scattered or diffuse area - details of iris clearly visible1 Easily discernible translucent areas, details of iris slightlyobscured 2 Opalescent areas, no details of iris visible, size of pupilbarely 3 discernible Opaque, iris invisible 4 B. Area of cornea involvedOne quarter (or less) but not zero 1 Greater than one quarter but lessthan one-half 2 Greater than one-half but less than three quarters 3Greater than three quarters up to whole area 4 Score equals A × B × 5Total maximum = 80 Iris A. Values Folds above normal, congestion,swelling, circumcorneal injection 1 (any one or all of these orcombination of any thereof), iris still reacting to light (sluggishreaction is positive) No reaction to light, hemorrhage; grossdestruction (any one or all 2 of these) Score equals A × 5 Totalpossible maximum = 10 Conjunctiva A. Redness (refers to palpebralconjunctiva only) Vessels definitely injected above normal 1 Morediffuse, deeper crimson red, individual vessels not easily 2 discernibleDiffuse beefy red 3 B. Chemosis Any swelling above normal (includesnictitating membrane) 1 Obvious swelling with partial eversion of thelids 2 Swelling with lids about half closed 3 Swelling with lids abouthalf closed to completely closed 4 C. Discharge Any amount differentfrom normal (does not include small amount 1 observed in inner canthusof normal rabbits) Discharge with moistening of the lids and hairs justadjacent to 2 the lids Discharge with moistening of the lids andconsiderable area around 3 the eye Score equals (A + B + C) × 2 Totalmaximum = 20 Draize et al. (1944) Methods for the study of irritationand toxicity of substances applied topically to the skin and mucousmembranes Scores of 0 are assigned for each parameter if the cornea,iris, or conjunctiva, are normal.

EXAMPLES

Compound-I is a potent and selective small molecule inhibitor ofVEGFR-2, along with other proangiogenic RTKs such as the FGF receptors(FGFR-1-3), Tie-2, and the ephrin receptor B4 (EPHB-4). Compound-I wasshown to inhibit phosphorylation of specific RTKs, endothelial cellproliferation, and pathologic angiogenesis following systemicadministration in murine cornea and rat growth plate models, as well asthe growth of human tumor xenographs in athymic mice. Regardingpotential ophthalmic indications, the Examples of the presentapplication described below demonstrated that topical ocular delivery ofCompound-I provided significant inhibition of pathologic retinal andchoroidal neovascularization in clinically-relevant rodent models. Asummary of these data follows.

The following studies were conducted to measure the effect of thedisclosed compounds on vascular leak and neovascularization of retinatissue.

Example 1

Primary Pharmacodynamics

In Vitro Efficacy Pharmacology of Compound-I.

Compound-I potently inhibits the tyrosine kinase activity of vascularendothelial growth factor receptor-2 (VEGF-2), as well as a selectsubset of other proangiogenic RTKs, during various in vitro assays.Specifically, Compound-I compound blocked VEGF-stimulated VEGFR-2phosphorylation in whole cells along with the proliferation of culturedendothelial cells. Compound-I inhibited recombinant tyrosine kinaseactivity of VEGFR-2 and FGFR-2 with a 50% inhibitory concentration(IC₅₀) of 10.55 nM (6 ng/mL) and 8.79 nM (5 ng/mL), respectively; andinhibited VEGFR-2 autophosphorylation in intact cells with an IC₅₀=5.27nM (3 ng/mL). This inhibition was selective versus many other tyrosinekinases, e.g., the VEGFR-2 IC₅₀ was approximately 500× and 1000× lowerthan those for epidermal growth factor receptor (EGFR) and the insulinreceptor (IR) tyrosine kinases, respectively (see Table 2).

When using a 10-point titration curve that ranged from 257 to 5000 nM(146-2845 ng/mL), Compound-I exhibited potent inhibition of tyrosinekinase activity for several proangiogenic growth factor receptors, asevidenced by an IC₅₀<100 nM (56.89 ng/mL) (see Table 3). The IC_(50s)for this select group of kinases were as follows: recombinant KDR (humanisoform of VEGFR-2)=1.27 nM (0.72 ng/mL), Tie-2=10.10 nM (5.75 ng/mL),and FGFRs 1-3=8.50 nM (4.84 ng/mL), 3.08 nM (1.75 ng/mL), and 33.9 nM(19.29 ng/mL), respectively. The compound also blocked the otherhigh-affinity VEGF receptor, VEGFR-1/Flt-1, but with lower potency:IC₅₀=122 nM (69.41 ng/mL).

Although VEGFR inhibition appears to be essential for reducing vascularpermeability and preventing further neovascular growth, the simultaneousinhibition of VEGF signaling with inhibition of other growth factorsignaling pathways (e.g., PDGF and angiopoietins/Tie2) may be linked tounique therapeutic outcomes. The therapeutic outcomes of a broaderinhibition of signaling pathways may contribute to the regression ofnewly established pathologic vessels in the posterior segment of theeye.

300 nM (170.67 ng/mL) of Compound-I completely inhibited VEGFR-2 kinasefunction (see Table 4) and provided substantial blockade of a similarset of proangiogenic growth factor receptors, including FGFRs-1-3,Tie-2, and EphB-4. An unexpected finding was that the 300 nMconcentration was able to completely inhibit the VEGFR-2 kinasefunction. This concentration falls within the typical range found in thecentral choroid and retina following five days of topical oculardelivery in rabbits and dogs.

Overview of Drug Substance and Drug Product

Drug Substance: The active pharmaceutical ingredient (API), Formula IIHydrochloride (Compound-I, CP-547,632-01), is a small molecule of asingle polymorph. The API substance is consistently manufactured inpurity exceeding 99.7%. Any impurity in drug substance ≧0.15% issuitably qualified in toxicology studies and the current specificationfor new unknown individual impurities is set to NMT 0.2%. The final drugsubstance and drug product are analyzed using standard methods.

Drug Product: Compound-I ophthalmic formulations for clinical studieswas manufactured in dosage strengths between 0.05%-1.0% (as Compound-I).The strengths used for the GLP batches are 0% (placebo), 0.05%, 0.1%,0.2%, 0.3%, 0.4%, 0.6%, 0.8%, and 1.0% Compound-I. Compound-I ophthalmicformulations (solutions or suspensions) are used for daily, single use,topical administration to the eye in the clinical trial. In addition tothe active ingredient, the drug product may further contain 0.005% BAKas a preservative, purified water as vehicle, and is pH-adjusted withsodium hydroxide to pH 6.0.

Example 2

Sodium Phosphate-Based Gel Drop

The ophthalmic benefits of Compound-I in a sodium phosphate-basedformulation (listed in Table 6) results from the self-gelling propertiesof the API in buffers, such as sodium phosphate. Spontaneous formationof a self-forming, thixotropic gel of Compound-I from a clear solutionwas formed by increasing API concentration in sodium phosphate. This gelinitially appeared clear and then demonstrated increasedthickness/viscosity at higher API concentrations, as well as becomingincreasingly more opaque, i.e., turbid. Once the API concentration inthe phosphate buffer reached super-saturated state, insolubleparticulates of Compound-I also were observed within the gel.

Application of a gel with increased viscosity to the surface of the eyeincreases corneal residence time. Increased corneal residence time inturn facilitates ocular drug absorption. As a result, the intraoculardrug concentrations of viscous gels increase in comparison tonon-viscous formulations, such as water-like solutions. One way toincrease viscosity is to use various viscosity-enhancing excipients,e.g., carboxymethylcellulose, which in effect achieves increasedintraocular absorption of different drug substances following topicalocular administration. In this study, however, a thixotropic gel ofCompound-I was unexpectedly formed in the absence of anyviscosity-enhancing excipients. For example, when Compound-I wasdissolved into a simple buffer, such as sodium phosphate, a thixotropicgel was formed. The thixotropic gel, which was formed without anyviscosity-enhancing excipients, was formulated as a Gel Drop in thisExample.

The Gel Drop of Compound-I was applied to eyes of Dutch-belted rabbits.Gel Drops of Compound-I were administered to Dutch-Belted rabbits for 4or 5 consecutive days, with three times daily dosing. The concentrationsof Compound-I at the target tissues were measured at 1 hour followingthe last administered dose. Delivery of Compound-I to the posteriorsegment tissues was dose-dependent and dose-frequency dependent.

The Gel Drop formulations (listed in Table 6) differed in severalaspects, such as API concentration, sodium phosphate concentration,presence or absence of tonicity (glycerin) or preservative(benzalkoniumchloride/BAK) agents, solubilizing surfactants (polysorbate80, tyloxapol, and/or poloxamer), and pH.

Example 3

Tromethamine-Based Suspension

Compound-I (about 1 mg/mL to about 10 mg/mL) in a tromethamine-basedformulation formed a suspension. The suspension of Compound-I in atromethamine-based formulation had >95% of the active drug substance inan insoluble form. This characteristic is distinguishable from thesoluble or semi-soluble state of Compound-I in the Gel Drop (the GelDrop (gel) is not an entirely soluble state as concentration of theactive agent increases) or in a Cyclodextrin-based formulation.Tromethamine-based formulations of Compound-I showed increased turbiditywith increasing active agent concentration. Administering a topical dropof Compound-I suspension to the eye (which is a combination of solubleand insoluble active agent components) was expected to provide uniquebenefits relevant to both safety/tolerability and efficacy.

Tromethamine-based suspension of Compound-I was administered toDutch-Belted rabbits for 4 or 5 consecutive days with three times dailydosing. Ocular tissue and plasma concentrations of Compound-I weremeasured at 1 hour following the last administered dose. Compound-I inthe tromethamine-based suspension delivered concentrations to the targettissues between 10-1000× of its cellular IC₅₀ for the variouspro-angiogenic RTKs. See Table 7.

The corneal safety and tolerability of topical Compound-I was a directconsequence of the amount of soluble (as opposed to insoluble) activeagent applied to the corneal surface, and the resultant corneal tissueconcentration. Animals that received topical ocular administration ofthe tromethamine-based suspension were able to tolerate up to higherlevel of the active agent concentration in the formulation, as comparedto equimolar formulations of the sodium phosphate-based Gel Drop.Results obtained from both Dutch-belted rabbits and beagle dogssuggested that ocular side effects, such as discomfort and inflammationand in some cases, corneal thinning, were more consistently observedwhen the cornea concentration of Compound-I exceeded 100 μM.

Administration of the tromethamine-based suspension had the unexpectedeffect on the ocular bioavailability of Compound-I in the posteriorsegment. The ocular bioavailability of Compound-I in the posteriorsegment was observed to be directly proportional to the total amount ofdrug administered (insoluble plus soluble, see Table 7). Although theinsoluble drug particulates were not readily available to anteriorsegment tissues; the inherent and unique physicochemical properties ofCompound-I allowed both insoluble and soluble components to gain entryto posterior segment tissues, such as the choroid and retina.Consequently, even higher drug concentrations than those achieved withGel Drop formulations containing equivalent amounts of the active agentwere achieved with the tromethamine-based suspension. Thus,tromethamine-based suspension provided: a) improved corneal tolerabilityand b) increased bioavailability to the posterior segment, particularlyto the choroid, the primary target tissue for treating neovascular (wet)AMD.

Example 4

Cyclodextrin-Based Solution

Cyclodextrins, which are cyclic oligosaccharides made up of six to eightdextrose units (α-, β-, and γ-CDs) joined through one to four bonds, arewell-known for their ability to act as a solubilizing agent forrelatively insoluble drugs. See Stella & He, Cyclodextrins, Toxicol.Pathol., 36: 30-42 (2008).

A clinical formulation of Compound-I or its free base ophthalmicSolution in 2-hydroxypropyl-β-cyclodextrin (HP-β-CD, KLEPTOSE® HPB) atequal to or more than 1:6 molar ratio or Sulfobutylether-β-cyclodextrin(SBE-β-CD, CAPTISOL®) at P at equal to or more than 1:2 ratio providedsolubility with clinical dose strengths of 0.1-1.0% Compound-I.

Cyclodextrin-based solutions of Compound-I or its free base not onlyimproved solubility of the active agent into a uniform solution, but,upon topical ocular administration, also had a novel and previouslyunobserved characteristic of significantly increased therapeutic indexof the active agent at the posterior segment of the eye. The solutionreduced anterior segment exposure, thereby providing increasedconcentration of the active in the solution and increased deliveryfrequency, which maintained high posterior segment concentrations. Bothof these beneficial characteristics are related to the known property ofcyclodextrin to form hydrophilic complexes with hydrophobic drugs. SeeStella & He, Cyclodextrins, Toxicol. Pathol., 36: 30-42 (2008). Whenformulated with Compound-I or its free base, cyclodextrin formed aclear, colorless solution and exhibited water-like viscosity. Followingtopical ocular administration, the Compound-I/cyclodextrin complex hadthe appearance of being pharmacologically inactive and metabolicallyinert. The Compound-I/cyclodextrin complex conferred cornealtolerability until cyclodextrin spontaneously dissociated from theactive agent, thus making available high concentration of Compound-I atits intended site of action in the posterior segment of the eye, e.g.,choroid and retina.

During topical ocular dosing studies lasting from 1 to 30 days inDutch-belted rabbits, cyclodextrin-based solutions of Compound-Idemonstrated dramatically lowered corneal exposures compared to Gel Dropformulations (see Example 2) at similar drug concentrations. The use ofcyclodextrin-based solutions of Compound-I provided an approximate 10×reduction in corneal concentrations, as compared to dosing withequimolar formulations of the Gel Drop. Consequently, after 30 days oftopical ocular dosing of 0.6% Compound-I as a cyclodextrin-basedsolution, no untoward findings were attributed to test-article orvehicle. The cyclodextrin-based solution of Compound-I also achievedequal or significantly higher concentrations of drug within theposterior segment target tissues, such as at the central choroid and thecentral retina. The combined effects of decreasing corneal drug exposureso as to avoid poor ocular tolerability, while increasing posteriorsegment bioavailability so as to increase RTK inhibition, maysignificantly increase the therapeutic index and correspondingbenefit(s) experienced by patients.

For both suspension-based formulations (see Example 3) and thecyclodextrin formulations, the therapeutic window is expanded due tosignificantly reduced exposure (10-100× or 1-2 log reduction). Thereduced exposure improves corneal safety/tolerability, which allowshigher concentrations or frequency of dosing of Compound-I to beadministered topically. The higher concentrations enables the drug toachieve higher back of the eye target tissue concentrations, whichimproves the therapeutic efficacy of Compound-I.

This study demonstrated that in rabbits and dogs, topical ocular dosingof ophthalmic gel drops, was associated with high corneal tissueexposure (≧100 uM) and corresponding untoward observations in theanterior segment, such as discomfort, corneal and conjunctivalinflammation, corneal epithelial erosion and/or thinning anddegeneration. In contrast, repeated topical ocular dosing of Compound-Iophthalmic solution produced corneal exposure that are roughly 5 to10-fold lower than an equimolar dose of ophthalmic gel drops, and arefree of untoward clinical or histopathologic findings. Furthermore,topical ocular dosing with Compound-I ophthalmic solution achieved equalor higher target therapeutic exposure in the central choroid incomparison to an equimolar dose of the ophthalmic gel drop. Overall, thecombination of decreased corneal exposure and corresponding improvedocular tolerability, while simultaneously maintaining or promoting drugdelivery to the posterior segment target tissues, along with improvedphysicochemical stability, will provide greater benefit to patientscompared to the ophthalmic Gel Drop formulation.

Example 5

1- to 5-Day PK Results with Topical Ocular Compound-I inCyclodextrin-Based Solutions

Following topical ocular dose administration of various formulations anddosage regimens of Compound-I in Dutch Belted rabbits, ocularpharmacokinetics was investigated. Nine (9) different topical ocularformulations having three doses of Compound-I were administered eitheronce per day (q.d.) or twice per day (b.i.d.) for 1, 4, or 5 consecutivedays. The study design (see Tables 10A-B) consisted of seventy-two (72)rabbits each receiving a 30 μL bilateral topical ocular dose of one ofthree (3) Compound-I formulations, or vehicle formulation, using apositive displacement pipette.

The composition of each Compound-I formulation is described in Table 8A.All doses were administered within ±1 hour of the scheduled dose time.On day 1, Groups 1, 2, 4-6, 8, 10, 11, 13, 15, and 17 received one dose(q.d.) for either one (1) or four (4) days. On days 1 through 4, Groups3, 7, 9, 12, 14, and 16 received b.i.d. dosing approximately 8 hoursapart at 7:00 μM and 3:00 μM for four (4) days. Animals in Groups 18 and19 received b.i.d dosing of vehicle only formulations for five (5)consecutive days.

Ocular sampling occurred for Group 1 at 0.5, 1, 2, 4, 8, or 24 hourspost-dose relative to the day 1 dose. Ocular sampling occurred forGroups 2 and 6 at 1, 8, and 24 hours post-dose relative to the day 5morning dose. Ocular sampling for Groups 3, 7, 8, 11, and 13 occurred at1 and 24 hours post-dose relative to the day 5 morning dose. Ocularsampling for Groups 4, 9, 10, 12, 14, 15, 16, and 17 occurred at 1 hourpost-dose relative to the day 5 morning dose. Group 5 ocular samplingoccurred at 0.5, 1, 2, 4, 8, and 24 hours post-dose relative to the day1 dose. Animals in Groups 18 and 19 were followed only by clinicalobservations for five (5) days.

Aqueous humor, cornea, central and peripheral retina, and central andperipheral choroid samples were collected. Aqueous humor, cornea,central retina, and central choroid samples were then assayed.Peripheral retina and peripheral choroid samples were assayed only forGroups 1-8, 12, 14, and 16 per study protocol.

Rabbit aqueous humor, cornea, central and peripheral retina, and centraland peripheral choroid samples were analyzed. Calibration curves wereprepared in control matrix to determine the concentration of Compound-Iin the various tissues.

Example 6

5-Day PK Results with Topical Ocular Compound-I in Cyclodextrin-BasedSolutions

Following ocular dose administration of various dose regimens of topicalocular solutions of Compound-I containing hydroxypropyl-β-cyclodextrin(“HDβCD”) in Dutch-Belted rabbits, the ocular pharmacokinetics wascalculated. Nine (9) different topical ocular solutions having fourdifferent doses of Compound-I were administered either once per day(q.d.) or twice per day (b.i.d.) for either 4 or 5 consecutive days. Thestudy design consisted of forty (40) rabbits each receiving a 30 μLbilateral topical ocular dose of one of four (4) Compound-I dosagestrengths, using a positive displacement pipette.

All doses were administered with ±1 hour of the scheduled dose time,except on day 1 for Groups 1, 4, and 10. The first dose on day 1 wasadministered at 12:00 μM, and the second dose (for Groups 1 and 10) wasadministered approximately 4 hours after. This was due to delayedarrival of formulations. All other dosing for these groups was asscheduled. On day 1, Groups 4-8, and 11-12 received one dose (q.d.) forfour (4) days. On days 1 through 4, Groups 1-3, and 9-10 received b.i.d.dosing approximately 8 hours apart for four (4) days.

Ocular sampling occurred one hour following the first daily dose on day5 for all groups, except Groups 5b and 6b, where ocular samplingoccurred 24 hours after the first daily dose on day 4.

Blood samples for plasma collection were obtained just prior toscheduled euthanasia for all animals. Aqueous humor, cornea, central andperipheral retina, and central and peripheral choroid samples werecollected. Cornea, central retina, and central choroid samples fromgroups 1-12 were assayed. Aqueous humor, peripheral retina, andperipheral choroid samples were not assayed.

Example 7

Concentrations of Compound-I (in μM) in Various Ocular Fluids andTissues

The ocular solution of Compound-I comprising cyclodextrin was preparedand tested in different groups of animals. Upon topical ocularadministration of a solution of 0.4% (4 mg/mL) Compound-I andcyclodextrin, the concentration of the active agent was measured invarious tissues and fluids of the eye. Average concentration ofCompound-I was measured in the central choroid, central retina, aqueoushumor, and cornea. Compound-I was in a solution (0.4% or 4 mg/mL) with8.41% KLEPTOSE® and 0.142% phosphate buffer; 8.9% KLEPTOSE® HPB and0.142% phosphate; 4.88% CAPTISOL® and 0.142% phosphate; or 4.88%CAPTISOL® and 0.122% phosphate. See Table 10A-B.

The central choroid concentration of Compound-I was between 0.259 μM and0.769 μM. See Table 10A. The central retina concentration of Compound-Iwas between 0.0531 μM-0.124 μM. See Table 10A. The aqueous humorconcentration of Compound-I was between 0.00313 μM-0.00656 μM. See Table10A. And the corneal concentration of Compound-I was 6.49 μM-30 μM. SeeTable 10A. The cyclodextrins used to prepare the solutions wereKLEPTOSE® HPB or CAPTISOL®. See Table 10B.

Example 8

Ocular Toxicology Studies

Dose-limiting ocular toxicity was characterized by corneal andconjunctival findings in Dutch-Belted rabbits and beagle dogs. Theseocular findings from repeat-dose toxicology studies with Compound-Iophthalmic gel drops were based upon clinical ophthalmic andhistopathologic evaluations and limited to conjunctival hyperemia,chemosis, congestion, and discharge, corneal opacification andepithelial erosion, and keratoconjunctivitis. No untoward alterationsinvolving deeper structures of the eye (iris, lens, ciliary body,retina, choroid, sclera) or the optic nerve were observed. Retinalfunction was normal in all test article and vehicle treated groupsduring full-field electroretinograms performed in rabbits.

The objectives for the exploratory ocular toxicology studies were toidentify: a) a topical ocular formulation that was well tolerated and b)one that could achieve the targeted therapeutic concentrations ofCompound-I in the central choroid.

Mean Compound-I ocular tissue concentrations following twice dailytopical dosing with 0.3% Compound-I ophthalmic gel drop formulationswith and without benzylalkonium chloride were highest in the cornea(236-260 μM)>>peripheral choroid (2.79-4.10 μM), central choroid(0.340-0.496 μM), peripheral retina (0.150-0.309 μM) and aqueous humor(0.0197-0.0395 μM) in Dutch-Belted rabbits.

Tris-based suspension formulations were well tolerated, where clinicalophthalmic examinations revealed a notable absence of corneal findingswith only a few sporadic incidences of mild conjunctivitis inDutch-Belted rabbits. Moreover, the eyes from animals that had received0.3% w/v Compound-I Tris-based suspension twice daily for 30 days wereconsidered normal during microscopic evaluations. Mean Compound-I oculartissue concentrations, assessed at 1 hour±15 minutes after the firstdaily topical ocular dose on day 30 for the twice daily topical dosingwith 0.3% Compound-I Tris-based suspensions with and withoutbenzylalkonium chloride, were highest in the cornea (2.69-3.10μM)>>peripheral choroid (0.781-1.21 μM), central choroid (0.303-0.319μM), peripheral retina (0.0819-0.0868 μM), central retina (0.0495-0.0592μM), and aqueous humor (0.00127-0.00145 μM).

Cyclodextrin-based solutions, using hydroxypropyl-beta-cyclodextrin(HP-β-CD, KLEPTOSE® HPB), were well tolerated when administeredtopically for 30 days, twice daily at 0.1% Compound-I (2.1% HP-β-CD),twice daily at 0.2% Compound-I (4.21% HP-β-CD), once or twice daily at0.4% Compound-I (8.41% HP-β-CD), and once or twice daily at 0.6%Compound-I (up to 12.62% HP-β-CD) in Dutch-Belted rabbits. Moreover, ina similar repeat dosing study, cyclodextrin-based solutions of 0.4% w/vCompound-I in KLEPTOSE® HPB, KLEPTOSE® HP, or CAPTISOL® werewell-tolerated when dosed twice daily for 24 days. No overt oculartoxicity related to Compound-I or vehicle treatment was found duringclinical ophthalmic or microscopic examinations in either study.

In the 24-day study, ocular tissue concentrations from eyes treated withcyclodextrin-based solutions of Compound-I were assessed at 1 hour±15minutes after the first daily topical ocular dose on day 24 were indescending order highest in the cornea (6.49-30 μM)>>center-punchchoroid (0.212-0.769 μM)>center-punch retina (0.0531-0.124)>aqueoushumor (0.002-0.007).

In summary, dose-limiting corneal toxicity was observed with Compound-Iophthalmic Gel Drop formulations. The ophthalmic Gel Drop rendersfive-fold to fifteen-fold higher corneal concentrations of Compound-Icompared to cyclodextrin based solution, and fifty-fold to hundred-foldhigher corneal concentrations of Compound-I compared to Tris-basedsuspensions. Compound-I Tris-based suspensions and cyclodextrin-basedsolutions were well tolerated with no evidence of overt ocular toxicity.Dose levels that were well tolerated for the cyclodextrin-basedsolutions or Tris-based suspensions of Compound-I when administered onceor twice daily ranged from about 0.005% to about 5.0% w/v for at least30 days. Cyclodextrin-based solutions also provided the highest centralchoroid concentrations of Compound-I when using equimolar doses of thethree formulations, and met or exceeded target therapeuticconcentrations.

Example 9

Phase I Protocol for Dose-Escalation Study in Patients with NeovascularAMD

The Phase I study is a twelve-week, open-label, dose-escalating,multi-center trial designed to evaluate the safety, tolerability, andpharmacokinetics following topical ocular administration of Compound-Iin patients with neovascular age-related macular degeneration (AMD). Upto 60 patients total are treated one to two times daily with topicalocular dosing of Compound-I ophthalmic solution for three months, wherethree dose-escalating monotherapy arms and one adjunct therapy arm usinga single intravitreal injection of LUCENTIS® plus themaximally-tolerated monotherapy dose are planned (15 patients pertreatment arm). Patients that meet pre-specified vision and CNV lesioncriteria confirmed by an independent reading center are allowed tosimultaneously discontinue topical ocular dosing and receive treatmentwith standard-of-care.

Compound-I ophthalmic solution for clinical studies is manufactured inat least 3 dosage strengths, ranging from 0.1% to 1.0% (as Compound-I).The strengths for the GLP batches are 0% (placebo), 0.1%, 0.3%, 0.6%,and 1.0% Compound-I HCl. Up to 2- to 3-fold incremental doses(approximately ½ log unit steps) are administered to succeeding cohorts.

Example 10

A “non-gel,” “non-viscous,” homogeneous ophthalmic solution topicalformulation that is both physically and chemically stable over the drugstrengths of 0.1-1.0% (1 to 10 mg/ml) was prepared by measuring theCompound-I concentration, cyclodextrin complexing agent concentration,pH, and tonicity, on Compound-I solubility and stability. A suitablebuffering system prevents pH drift on stability at concentrations lessthan 1 mg/mL. Both phosphate and Trometamol (Tris) were evaluated asbuffering agents. Sodium chloride was used to adjust tonicity.

The product quality attributes are shown in Table 14.

TABLE 14 Product Quality Attributes Solvent Solubility (mg/mL) ColorAppearance Clear colorless with no visually apparent of formulation pHpH 5.5-7.0 Turbidity Clear Viscosity Free flowing, water-like andfilterable Tonicity Isotonic Sedimentation None Mixing end point Clearcolorless with no visually apparent Solubility Solubility ≧6 mg/mlFormulation Preparation

The formulations outlined in Tables 12 and 13 were prepared using thegeneral procedure listed.

The formulation was made up to volume with water for injection andstirred for 30 minutes at 500 rpm. Final pH was checked and adjustedwith either, NaOH or HCl to the target range. Approximately 5 mlaliquots is directly filtered into semi-transparent 5 ml LDPE bottleswhile continuously stirring at constant speed with the aid of WatsonMarlow Pumpsil D tubing, fitted to a Flexicon filler and attached to 0.2micron PVDF capsule filter. Samples are stored at 2-8° C. until allsample preparation is complete. All samples will then be submitted toanalytical for storage and testing.

Example 11

EGFR Tyrosine Phosphorylation Assay in Corneal Epithelial Cells (hTCEpiCells) to Determine EGFR Activity of Compounds of Formula I or II

An EGFR tyrosine assay in corneal epithelial cells was performed todetermine whether higher concentrations of EGF can overcome inhibitionof EGFR kinase activity by a compound of Formula I or II.

35 mm dishes of hTCEpi cells were serum starved and then werepre-treated with different concentrations of a compound of Formula I orII, e.g., Compound-I, or a control, AG1478 (an EGFR kinase inhibitor),for 30 minutes, followed by treatment with EGF (10, 50, 100 ng/ml) for15 minutes. Six concentrations of a compound of Formula I or II, e.g.,0, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM, or 1 μM AG1478 as a control, wereused for each EGF concentration (10, 50, 100 ng/ml). Additional controlsrun were no compound of Formula I or II or no EGF. All experiments wererepeated three times. Cells were then harvested and immunoblotted fordetermination of phosphorylated EGFR (tyrosine 1068 and tyrosine 1045)and total EGFR.

EGFR phosphorylation serves as a readout of receptor activity. As shownin FIG. 1, high concentrations of a compound of Formula I or II wereable to inhibit ligand-stimulated EGFR tyrosine phosphorylation, andincreasing concentrations of a compound of Formula I or II decreasedEGFR tyrosine phosphorylation at both sites. Increasing concentrationsof EGF overcome the EGFR inhibition and corneal adversity due to thecompound of Formula I or II (FIG. 1). The EGFR-specific inhibitor,AG1478, has an IC₅₀=3 nM, and at 1 μM AG1478 completely blocked EGFRphosphorylation. It is estimated that 100 μM of a compound of Formula Ior II inhibits EGFR phosphorylation ˜75-80%.

Example 12

EGFR Tyrosinephosphorylation Assay in Corneal Epithelial Cells (hTCEpiCells) with Compounds of Formula I or II, and a Second Active Agent

An EGFR tyrosine assay in corneal epithelial cells was performed todetermine if vitamin K, nicotinic acid, or nicotinamide can preventcompounds of Formula I or II from inhibiting EGFR and if there is anincrease in EGFR activity.

35 mm dishes of hTCEpi cells were serum starved and then werepre-treated with vitamin K (50 μM), nicotinic acid (10 μM), ornicotinamide (10 μM) for 4 hours, followed by treatment with differentconcentrations of a compound of Formula I or II, e.g., Compound-I, for30 minutes. The cells were then incubated with EGF (50 ng/ml) for 15minutes. Cells were harvested and cell lysates were immunoblotted fordetermination of phosphorylated EGFR (tyrosine 1068 and tyrosine 1045)and total EGFR concentration. As shown in FIG. 2, vitamin K, nicotinicacid, or nicotinamide overcome Formula I or II compound-mediated cornealadversity (EGFR inhibition). In addition, FIG. 2 shows that vitamin K₃(menadione) most effectively attenuated Formula I or IIcompound-mediated inhibition of EGFR tyrosine phosphorylation.

Example 13

Determination of Cell Migration/Proliferation (In Vitro Wound Healing)in Corneal Epithelial Cells (hTCEpi Cells): Effects of Compounds ofFormula I or II and EGF

Determination of cell migration/proliferation in corneal epithelialcells was performed to determine cell migration/proliferation in thepresence of varying concentrations of a compound of Formula I or II andEGF.

hTCEpi cells were plated with silicone plugs. The cells were then serumstarved and pre-treated with varying concentrations of a compound ofFormula I or II or a control compound, AG1478, for 30 minutes. Thesilicone plugs were then removed to create the acellular area. The cellswere photographed and then treated with EGF (10, 50, or 100 ng/ml) orVEGF (10 ng/ml) together with a compound of Formula I or II or AG1478for 16 hours. The cells were again photographed and cell migration wasquantified from the micrographs. All experiments were repeated threetimes.

Four concentrations of a compound of Formula I or II (0, 3 μM, 10 μM, 30μM) or 3.2 μM AG1478 (an EGFR kinase inhibitor), were used for each EGFconcentration or VEGF. Additional controls run included no compound ofFormula I or II and no EGF.

The data allows determination of whether a compound of Formula I or IIprevents the in vitro measures of corneal epithelial wound healing (cellmigration and proliferation) and if higher levels of EGF overcomereceptor tyrosine kinase inhibition. As shown in FIGS. 3A and 3B, acompound of Formula I or II caused a dose-dependent inhibition ofEGFR-mediated hTCEpi cell in vitro “wound healing”.

Example 14

Determination of Cell Migration/Proliferation (In Vitro Wound Healing)in Corneal Epithelial Cells (hTCEpi Cells): Effects of Compounds ofFormula I or II and a Second Active Agent

Determination of cell migration/proliferation in corneal epithelialcells was performed to determine cell migration/proliferation in thepresence of varying concentrations of a compound of Formula I or IIalone or together with vitamin K, nicotinic acid, or nicotinamide.

hTCEpi cells were plated with silicone plugs. The cells were then serumstarved and pre-treated with varying concentrations of vitamin K₃analog, menadione, for 4 hours and supplemented with the varyingconcentrations of a compound of Formula I or II for 30 minutes. Theplugs were removed. The cells were photographed and then treated withvarious concentrations of vitamin K, EGF, and/or a compound of Formula Ior II. The cells were again photographed and cell migration wasquantified from the micrographs. Additional controls run included nocompound of Formula I or II and no EGF. All experiments were repeatedthree times.

As shown in FIGS. 4A and 4B, the inhibitory effects of a compound ofFormula I or II on in vitro wound healing can be reversed by theaddition of 0.3 μM menadione. There was a statistically significantdifference in in vitro wound healing when 0.3 μM of a compound ofFormula I or II was combined with 0.3 μM menadione versus 0.3 μM of acompound of Formula I or II alone.

Example 15

Determination of Basal and EGF-Mediated Corneal Wound Healing In Vivo:Effects of Compounds of Formula I or II

Determination of the effects of a compound of Formula I or II on basaland ligand stimulated rates of corneal wound healing was determined inC57/Bl mice.

Corneas of 8 week old C57/Bl mice were wounded (1.5 mm superficialepithelial wound) and then pre-treated with a compound of Formula I orII (0, 1.0 μM, 10 μM, 30 μM, or 50 μM), followed by addition of EGF (0,10, 100 ng/ml). As a control, AG1478 (an EGFR kinase inhibitor) was usedin the absence and presence of 100 ng/ml EGF. Wound size was monitoredby fluorescein staining and fluorescent photography over the course of40 hours. Wound closure was quantified using Image J software.

As shown in FIGS. 5A, 5B, 5C, and 5D, in vivo corneal epithelial woundhealing was reduced in the presence of a compound of Formula I or II ina dose dependent manner. Maximal inhibition of wound healing wasobserved with a compound of Formula I or II concentrations that werefive times greater than those of AG1478. Pre-treatment with a compoundof Formula I or II for 48 hours before making the wound to the corneainhibited corneal epithelial wound healing at a lower concentration of acompound of Formula I or II than without the pre-treatment.

Example 16

Determination of Basal and EGF-Mediated Corneal Wound Healing In Vivo:Effects of Compound of Formula I or II and a Second Active Agent

Determination of the effects of a compound of Formula I or II and/or asecond active agent on corneal wound healing was determined in C57/Blmice.

Corneas of 8 week old C57/Bl mice were pre-treated with topicaladministration of vehicle, 0.3 μM menadione, 10 μM of a compound ofFormula I or II, 0.3 μM menadione and 10 μM of a compound of Formula Ior II 48 hours before the corneas were wounded (1.5 mm superficialepithelial wound). Wound size was monitored by fluorescein staining andfluorescent photography. Wound closure was monitored over the course of40 hours.

As shown in FIGS. 6A, 6B, and 6C, menadione (0.3 μM) reversed theeffects of a compound of Formula I or II (10 μM) pre-treatment oncorneal epithelial wound healing.

Example 17

Effect of Menadione on Inhibition of VEGFR by Compounds of Formula I orII

Human retinal endothelial cells were treated with 0, 0.3, or 3 μMmenadione for 4 hours, followed by 30 minutes of treatment with varyingconcentrations of a compound of Formula I or II (1 nM, 10 nM, 100 nM, or1 μM) in menadione. The cells were then stimulated with 0.5 nM (10ng/ml) VEGF. The cells were lysed. The lysates were resolved by 7.5%SDS-PAGE, transferred to nitrocellulose, and immunoblotted forphosphorylated VEGFR2, total VEGFR2, or α-tubulin as a loading control.As shown in FIG. 7A, menadione did not change the IC₅₀ of inhibition ofVEGFR2 phosphorylation mediated by a compound of Formula I or II.

Example 18

Effect of Menadione on the Proliferation of Primary Human RetinalMicrovascular Endothelial Cells

Human retinal endothelial cells were pretreated with menadione for 4hours, then with the indicated concentration of a compound of Formula Ior II for 30 minutes, and supplemented with 0.5 nM (10 ng/ml) VEGFovernight. Viable cells were quantified by Alamar Blue Assay (ThermoFisher). Data were plated as the fold growth relative to cells treatedwith no VEGF or a compound of Formula I or II, and are shown as theaverage±S.E.M. in FIG. 7B. As shown in FIG. 7B, menadione did not changethe IC₅₀ of inhibition of VEGF-mediated retinal endothelial cellproliferation mediated by a compound of Formula I or II.

Example 19

Determination of Mechanism by which Menadione Enhances EGFR Activity

hTCEpi cells were pretreated with menadione (0, 0.3, or 3.0 μM) for 4hours. Cells were then incubated with 8.0 nM (50 ng/ml) EGF for 0-3hours. The cells were lysed and the lysates were resolved by SDS-PAGE,and immunoblotted for phosphorylated EGFR (pY1068), total EGFR, orα-tubulin. As shown in FIG. 8, menadione treatment did not change basalEGFR phosphorylation or slow EGFR dephosphorylation. 0.3 mM menadioneslowed the kinetics of EGFR degradation, indicating that menadione maysustain EGFR receptor signaling by slowing the kinetics of receptordegradation or increasing the rate of new receptor synthesis.

Example 20-1

Ocular Pharmacokinetics of Formulations of the Application in DutchBelted Rabbits

A non-GLP study was conducted to assess the ocular pharmacokinetics of afirst active agent of the present application (e.g., Formula II orCompound-I), in suspension or solution, when administered once or twicedaily, as a topical instillation, to both eyes of Dutch Belted rabbitsfor four days. The study design (Table 15-1) consisted of forty-five(45) Dutch Belted rabbits, each receiving a 30 μL bilateral topicalocular dose. The administration was conducted according to the dosingregimen in Table 15-1.

TABLE 15-1 Study design Dosing No. of Dose Frequency Sample GroupRabbits Test Article Formulation Conc. Volume and Duration^(a) TimePoints 1 A 3 0.4% Formula II; 4 30 μL/ QD for 4 1 hr post dose 0.08%Pluronic F-127; mg/mL eye/dose Days on Day 5 2.5% Glycerol; LargeParticles in suspension 2 B 3 0.4% Formula II; 4 30 μL/ QD for 4 1 hrpost dose 0.08% Pluronic F-127; mg/mL eye/dose Days on Day 5 2.5%Glycerol; Nano Particles in suspension 3 C 3 0.2% Formula II; 2 30 μL/QD for 4 1 hr post dose 0.04% Pluronic F-127; mg/mL eye/dose Days on Day5 2.5% Glycerol; Large Particles in suspension 4 D 3 0.2% Formula II; 230 μL/ QD for 4 1 hr post dose 0.04% Pluronic F-127; mg/mL eye/dose Dayson Day 5 2.5% Glycerol; Nano Particles in suspension 5 E 3 Compound-I 430 μL/ QD for 4 1 hr post dose Ophthalmic solution mg/mL eye/dose Dayson Day 5 6 F 3 Compound-I 2 30 μL/ QD for 4 1 hr post dose Ophthalmicsolution mg/mL eye/dose Days on Day 5 7 G 3 Compound-I 1 30 μL/ QD for 41 hr post dose Ophthalmic solution mg/mL eye/dose Days on Day 5 8 H 3Compound-I 0.1 30 μL/ QD for 4 1 hr post dose Ophthalmic solution mg/mLeye/dose Days on Day 5 9 I 3 0.4% Formula II; 4 30 μL/ BID for 4 1 hrpost dose 0.08% Pluronic F-127; mg/mL eye/dose Days on Day 5 2.5%Glycerol; Large Particles in suspension 10 J 3 0.4% Formula II; 4 30 μL/BID for 4 1 hr post dose 0.08% Pluronic F-127; mg/mL eye/dose Days onDay 5 2.5% Glycerol; Nano Particles in suspension 11 K 3 0.2% FormulaII; 2 30 μL/ BID for 4 1 hr post dose 0.04% Pluronic F-127; mg/mLeye/dose Days on Day 5 2.5% Glycerol; Large Particles in suspension 12 L3 0.2% Formula II; 2 30 μL/ BID for 4 1 hr post dose 0.04% PluronicF-127; mg/mL eye/dose Days on Day 5 2.5% Glycerol; Nano Particles insuspension 13 M 3 Formula II 2 30 μL/ BID for 4 1 hr post doseOphthalmic solution mg/mL eye/dose Days on Day 5 14 N 3 Formula II 1 30μL/ BID for 4 1 hr post dose Ophthalmic solution mg/mL eye/dose Days onDay 5 15 O 3 Formula II 0.1 30 μL/ BID for 4 1 hr post dose Ophthalmicsolution mg/mL eye/dose Days on Day 5 ^(a)A single dose will beadministered on Day 5. OQ - once daily; BID - twice daily

Ocular sampling occurred one hour following the first daily dose on Day5 for all groups. Whole blood samples using K₂EDTA as an anticoagulantwere collected from all animals on Day 5, 1 hour post dose. The wholeblood was placed on ice until samples were spun in a centrifuge toseparate the plasma. Aqueous humor, conjunctiva, cornea, central retina,peripheral retina, central choroid, and peripheral choroid samples werecollected following necropsy. Samples were then frozen at −70° C. orlower. All Groups 1-15 plasma, central retina, central choroid, andcornea samples were assayed.

For all dose groups, ocular tissue concentrations of Formula II orCompound-I assessed on Day 5 were in descending order: highest in thecornea>>central choroid>central retina (Table 15-2). The highest corneaconcentrations of Formula II or Compound-I were seen in Group 10. Thelowest cornea concentrations of Formula II or Compound-I were seen inGroup 8. In general, higher central retina and central choroidconcentrations were associated with higher cornea concentrations ofFormula II or Compound-I. No advantage of other formulations overophthalmic solutions of Formula II or Compound-I was seen with respectto a reduction in cornea concentrations or an increase in central retinaor central choroid concentrations of Formula II or Compound-I.

TABLE 15-2 Plasma* Central Retina Central Choroid Cornea Group (μM) (μM)(μM) (μM) 1 0.00500 0.0560 0.152 22.1 2 0.00487 0.0620 0.117 34.0 30.00238 0.0312 0.127 9.25 4 0.00294 0.0353 0.108 21.4 5 0.00424 0.05510.116 11.9 6 0.00319 0.0413 0.101 9.60 7 0.00234 0.0258  0.0941 6.98 8<LLOQ <LLOQ <LLOQ 0.783 9 0.00350 0.0707 0.155 29.0 10 0.00391 0.09200.336 62.2 11 0.00226 0.0412 0.162 16.0 12 0.00308 0.0709 0.111 28.8 130.00256 0.0472 0.149 16.1 14 0.00214 0.0316 0.108 6.63 15 <LLOQ  0.00926<LLOQ 0.925 *Average based on n = 3 Plasma LLOQ = 0.00188 μM CentralRetina LLOQ = 0.00751 μM Central Choroid LLOQ = 0.0563 μM Cornea LLOQ =0.0376 μM

Gross ocular examinations using the Draize scale for scoring ocularirritation were performed on all animals prior to the first dose on eachday during the study. All formulations were well tolerated. In addition,animals were observed for general health pre-study, during dosing, andat necropsy. All animals assigned to study were normal. Necropsyevaluations consisted of observations in addition to those already notedon Draize Score sheets and Ophthalmologic Exam forms. For allformulations, dosing observations were assessed using the Draize ocularirritation scoring system. No abnormal observations were noted exceptfor the following: prior to necropsy on Day 5, Redness Level 1 in botheyes was noted in one animal in Group I and one animal in Group M.

Example 20-2

Ocular Pharmacokinetics of Formulations of the Application in DutchBelted Rabbits

A non-GLP study was conducted to assess the ocular pharmacokinetics of afirst active agent of the present application (e.g., Formula II orCompound-I), in suspension or solution, when administered one, three, orfour times daily, as a topical instillation, to both eyes of DutchBelted rabbits for four days. The study design (Table 16-1) consisted ofthirty-nine (39) Dutch Belted rabbits, each receiving a 30 μL bilateraltopical ocular dose. The administration was conducted according to thedosing regimen in Table 16-1.

TABLE 16-1 Study design Dosing No. of Dose Frequency Sample GroupRabbits Test Article Formulation Conc. Volume and Duration^(a) TimePoints 1 A 3 Compound-I 4 30 μL/ QD for 4 1 hr post dose OphthalmicSolution (Control) mg/mL eye/dose Days on Day 5 2 B 3 Compound-I 2 30μL/ QD for 4 1 hr post dose Ophthalmic Solution (Control) mg/mL eye/doseDays on Day 5 3 C 3 Compound-I 0.1 30 μL/ QD for 4 1 hr post doseOphthalmic Solution mg/mL eye/dose Days on Day 5 4 D 3 Compound-I 0.1 30μL/ TID for 4 1 hr post dose Ophthalmic Solution mg/mL eye/dose Days onDay 5 5 E 3 0.4% Formula II; 4 30 μL/ QD for 4 1 hr post dose Large 3 μmparticles in mg/mL eye/dose Days on Day 5 suspension 6 F 3 0.4% FormulaII; 4 30 μL/ QD for 4 1 hr post dose Large Particles, mg/mL eye/doseDays on Day 5 3 μm + HPBCD in suspension 7 G 3 0.4% Formula II; 4 30 μL/QD for 4 1 hr post dose Extra-large 35 μm mg/mL eye/dose Days on Day 5particles in suspension 8 H 3 0.2% Formula II; 2 30 μL/ QD for 4 1 hrpost dose Extra-large 35 μm mg/mL eye/dose Days on Day 5 particles insuspension 9 I 3 0.4% Formula II; 4 30 μL/ QD for 4 1 hr post doseExtra-large mg/mL eye/dose Days on Day 5 35 μm + HPBCD in suspension 10J 3 0.3% Compound-I; 3 30 μL/ QD for 4 1 hr post dose Tris; mg/mLeye/dose Days on Day 5 Particles in suspension 11 K 3 0.3% Compound-I; 330 μL/ QD for 4 1 hr post dose Tris + HPBCD; mg/mL eye/dose Days on Day5 Particles in suspension 12 L 3 0.4% Formula II; 4 30 μL/ QD for 4 1 hrpost dose HPMC; mg/mL eye/dose Days on Day 5 0.3 μM Vit K3; Extra-largeparticles in suspension 13 M 3 0.4% Formula II; 4 30 μL/ QD for 4 1 hrpost dose HPMC; mg/mL eye/dose Days on Day 5 1.0 μM Vit K3; Extra-largeparticles in suspension ^(a)A single dose will be administered on Day 5.QD - Once daily, TID - Three times daily, QID - Four times daily

Ocular sampling occurred one hour following the first daily dose on Day5 for all groups. Whole blood samples using K₂EDTA as an anticoagulantwere collected from all animals on Day 5, 1 hour post dose. The wholeblood was placed on ice until samples were spun in a centrifuge toseparate the plasma. Aqueous humor, conjunctiva, cornea, central retina,peripheral retina, central choroid, and peripheral choroid samples werecollected following necropsy. Samples were then frozen at −70° C. orlower. All Groups 1-13 plasma, central retina, central choroid, andcornea samples were assayed.

For all dosing groups, ocular tissue concentrations of Formula II orCompound-I assessed on Day 5 were in descending order highest: in thecornea>>central choroid>central retina (Table 16-2). The highest corneaconcentrations of Formula II or Compound-I were seen in Group 1. Thelowest cornea concentrations of Formula II or Compound-I were seen inGroup 3. In general, higher central retina and central choroidconcentrations were associated with higher cornea concentrations ofFormula II or Compound-I. No advantage of other formulations overFormula II or Compound-I ophthalmic solution was seen with respect to areduction in cornea concentrations or an increase in central retina orcentral choroid concentrations of Formula II or Compound-I.

TABLE 16-2 Plasma* Central Retina Central Choroid Cornea Group (μM) (μM)(μM) (μM) 1 0.00792 0.0858 0.170 16.4 2 0.00284 0.0387 0.0757 11.9 3<LLOQ 0.00868 0.0299 1.01 4 <LLOQ 0.00934 <LLOQ 1.18 5 0.00257 0.03460.0787 16.1 6 0.00451 0.0481 0.118 8.25 7 0.00223 0.0570 0.108 3.38 8<LLOQ 0.0208 0.0598 2.09 9 0.00280 0.0217 0.0581 3.43 10 0.00271 0.05970.195 3.90 11 0.00440 0.0532 0.145 3.98 12 0.00202 0.0204 0.0582 3.79 130.00193 0.0248 0.0700 3.08 *Average based on n = 3 Plasma LLOQ = 0.00188μM Central Retina LLOQ = 0.00751 μM Central Choroid LLOQ = 0.0282 μMCornea LLOQ = 0.0376 μM

Gross ocular examinations using the Draize scale for scoring ocularirritation were performed on all animals prior to the first dose on eachday during the study. All formulations were well tolerated. In addition,animals were observed for general health pre-study, during dosing, andat necropsy. All animals assigned to study were normal. Necropsyevaluations consisted of observations in addition to those already notedon Draize Score sheets and Ophthalmologic Exam forms. For allformulations, dosing observations were assessed using the Draize ocularirritation scoring system. The following abnormal observations werenoted throughout the duration of the study: animals in Groups 7, 9, 10,11, 12, and 13 experienced increased blinking following dosingthroughout various days of the study; one animal in Group 11 exhibitedDischarge Level 1 on Day 3.

Example 20-3

Ocular Pharmacokinetics of Formulations of the Application in DutchBelted Rabbits

A non-GLP study was conducted to assess the ocular pharmacokinetics of afirst active agent of the present application (e.g., Formula II orCompound-I), in suspension, when administered one, two, or three timesdaily, as a topical instillation, to both eyes of Dutch Belted rabbitsfor four days. The study design (Table 17-1) consisted of thirty-nine(39) Dutch Belted rabbits, each receiving a 30 μL bilateral topicalocular dose. Various suspension formulations of the present applicationwere evaluated and compared against two control formulations: a) acyclodextrin-based eye drops solution (Control Group 13, Table 17-1),and b) a pilot suspension formulation used in previous rabbitpharmacokinetic studies, (Group 10, Table 17-1). A comparison of theformulation variables for each suspension listed in Table 17-1 isprovided in Table 17-2. Formula II or Compound-I was used in the testsuspensions, whereas the Eye Drops solution formulation is derived withthe Compound-I.

TABLE 17-1 Study design Dosing No. of Dose Frequency Sample GroupRabbits Test Article Formulation Conc. Volume and Duration^(a) TimePoints 1 A 3 Formula II; 0.3 30 μL/ BID for 4 1 hr post dose Pluronic;mg/mL eye/dose Days on Day 5 35 μm particles 2 B 3 Formula II; 0.3 30μL/ TID for 4 1 hr post dose Pluronic; mg/mL eye/dose Days on Day 5 35μm particles 3 C 3 Formula II; 4 30 μL/ QD for 4 1 hr post dose HPMC;mg/mL eye/dose Days on Day 5 35 μm particles 4 D 3 Formula II; 4 30 μL/QD for 4 1 hr post dose Pluronic; mg/mL eye/dose Days on Day 5 35 μmparticles (Control) 5 E 3 Formula II; 4 30 μL/ QD for 4 1 hr post doseHPMC; mg/mL eye/dose Days on Day 5 50-60 μm particles 6 F 3 Formula II;4 30 μL/ QD for 4 1 hr post dose Pluronic; mg/mL eye/dose Days on Day 550-60 μm particles 7 G 3 Formula II; 4 30 μL/ QD for 4 1 hr post dosePVP; mg/mL eye/dose Days on Day 5 50-60 μm particles 8 H 3 Formula II; 430 μL/ QD for 4 1 hr post dose HPMC; mg/mL eye/dose Days on Day 5 1.0 μMVit K3; 50-60 μm particles 9 I 3 Formula II; 4 30 μL/ QD for 4 1 hr postdose Pluronic; mg/mL eye/dose Days on Day 5 1.0 μM Vit K3; 50-60 μmparticles 10 J 3 Compound-I; 4 30 μL/ QD for 4 1 hr post dose Tris;mg/mL eye/dose Days on Day 5 50-60 μm particles (Control) 11 K 3Compound-I; 4 30 μL/ QD for 4 1 hr post dose Tris + Tween80; mg/mLeye/dose Days on Day 5 50-60 μm particles 12 L 3 Compound-I; 4 30 μL/ QDfor 4 1 hr post dose Tris + HPMC; mg/mL eye/dose Days on Day 5 50-60 μmparticles 13 M 3 Compound-I; 4 30 μL/ QD for 4 1 hr post dose OphthalmicSolution mg/mL eye/dose Days on Day 5 ^(a)A single dose will beadministered on Day 5. QD - Once daily, BID - Twice daily, TID - Threetimes daily

TABLE 17-2 Group # 1 and 2 3 4 5 6 7 8 9 10 11 12 Dose- 0.3 4 4 4 4 4 44 4* 4* 4* strength mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mLmg/mL mg/mL mg/mL API Form Free Free Free Free Free Free Free Free HClHCl HCl Base Base Base Base Base Base Base Base Salt Salt Salt Particle35 35 35 50-60 50-60 50-60 35 35 50-60 50-60 50-60 size (μm) StabilizerPluronic HPMC Pluronic HPMC Pluronic PVP HPMC Pluronic Tris Tris +Tris + Tween 80 HPMC Vitamin — — — — — —  1  1 — — — K3 (μM) NOTES 2Xvolume QD QD QD QD QD QD QD QD QD QD for BID & (control) (control) TIDdosing frequencies *Note: requires 4.3 mg/mL of HCl salt to provide 4mg/mL of Free Base

Ocular sampling occurred one hour following the first daily dose on Day5 for all groups. Whole blood samples using K₂EDTA as an anticoagulantwere collected from all animals on Day 5, 1 hour post dose. The wholeblood was placed on ice until samples were spun in a centrifuge toseparate the plasma. Aqueous humor, conjunctiva, cornea, central retina,peripheral retina, central choroid, and peripheral choroid samples werecollected following necropsy. Samples were then frozen at −70° C. orlower. For all Groups 1-13, plasma, central retina, central choroid, andcornea samples were assayed.

Ocular tissue concentrations of Formula II or Compound-I assessed on Day5 were in descending order: highest in the cornea>>centralchoroid>central retina. Corneal concentrations of Formula II orCompound-I ranged from 1.5 to 14.7 μM with the highest concentrationsbeing observed in Group 13 and the lowest in Group 1. All suspensionformulations, Groups 1-12, provided lower average corneal concentrationsas compared to the Eye Drops solution control, Group 13. Theseobservations did not seem to be influenced by the presence or absence ofVitamin K₃/menadione in the suspension formulation. Central choroidconcentrations ranged from <0.028 to 0.27 μM with the highestconcentrations seen in Groups 11 and the lowest in Group 2. Centralretina concentrations ranged from 0.009 to 0.069 μM; Group 11 had thehighest central retina concentrations and Group 1 had the lowest centralretina concentrations. Plasma concentrations of Formula II or Compound-Iwere very low being approximately 10-fold and 100-fold lower than thosein the central retina and central choroid, respectively. See Table 17-3.

TABLE 17-3 Plasma* Central Retina Central Choroid Cornea Group (μM) (μM)(μM) (μM) 1 <LLOQ 0.00881 0.0319 1.50 2 <LLOQ 0.00884 <LLOQ 1.73 30.00205 0.0228 0.0767 4.38 4 0.00220 0.0305 0.0684 5.08 5 <LLOQ 0.02210.0558 3.31 6 <LLOQ 0.0279 0.101 4.82 7 0.00331 0.0282 0.0876 5.23 80.00257 0.0540 0.0844 4.05 9 0.00256 0.0344 0.0828 5.64 10 0.006230.0632 0.191 6.20 11 0.00410 0.0691 0.265 8.67 12 0.00470 0.0482 0.1165.82 13 0.00416 0.0570 0.112 14.7 *Average based on n = 3 Plasma LLOQ =0.00188 μM Central Retina LLOQ = 0.00751 μM Central Choroid LLOQ =0.0282 μM Cornea LLOQ = 0.0376 μM

Additionally, Vitamin K3 concentrations were determined in plasma,central retina, central choroid, and cornea samples for animals fromGroups 8 and 9. A calibration curve prepared in control matrix was usedto determine the concentration of Vitamin K3. See Table 17-4.

TABLE 17-4 Plasma* Central Retina Central Choroid Cornea Group (μM) (μM)(μM) (μM) 8 <LLOQ 15.4 <LLOQ <LLOQ 9 0.465 <LLOQ <LLOQ <LLOQ *Averagebased on n = 3 Plasma LLOQ = 0.291 μM Central Retina LLOQ = 11.6 μMCentral Choroid LLOQ = 43.6 μM Cornea LLOQ = 2.91 μM

Gross ocular examinations using the Draize scale for scoring ocularirritation were performed on all animals prior to the first dose on eachday during the study. All formulations were well tolerated, with theexception that for one animal in Group 11 on Day 4 of dosing, whereconjunctival chemosis was noted, and for one animal in Group 12 on Day 4of dosing, where conjunctival redness was noted. In addition, animalswere observed for general health pre-study, during dosing, and atnecropsy. All animals assigned to study were normal. Necropsyevaluations consisted of observations in addition to those already notedon Draize Score sheets. For all formulations, dosing observations wereassessed using the Draize ocular irritation scoring system. Thefollowing abnormal observations were noted throughout the duration ofthe study: animals in Groups 4, 6, 7, 8, 9, and 11 experienced increasedblinking following dosing throughout various days of the study; oneanimal in Group 4 exhibited Redness Level 1 in both eyes and ChemosisLevel 1 in the left eye prior to necropsy on Day 5; and one animal inGroup 5 exhibited Chemosis Level 1 in the right eye prior to necropsy onDay 5.

Example 20-5

Ocular Pharmacokinetics of Formulations of the Application in DutchBelted Rabbits

A non-GLP study was conducted to assess the ocular pharmacokinetics of afirst active agent of the present application (e.g., Formula II orCompound-I), in suspension, when administered one or two times daily, asa topical instillation, to both eyes of Dutch Belted rabbits for fourdays. The study design (Table 18-1) consisted of forty-nine (49) DutchBelted rabbits, each receiving a 30 μL bilateral topical ocular dose.Various suspension formulations of the present application wereevaluated and compared against a cyclodextrin-based eye drops solution(Control Groups 2 and 3, Table 18-1). A comparison of the formulationvariables for each suspension listed in Table 18-1 is provided in Table18-2. Formula II or Compound-I was used in the test suspensions, whereasthe Eye Drops solution formulation is derived with Compound-I.

TABLE 18-1 Dosing No. of Dose Frequency Sample Group Rabbits TestArticle Formulation Conc. Volume and Duration^(a) Time Points 1 A 3Compound-I; 0.3 30 μL/ BID for 4 1 hr post dose Ophthalmic Solution, pH6 mg/mL eye/dose Days on Day 5 2 B 4 Compound-I; 1 30 μL/ QD for 4 1 hrpost dose Ophthalmic Solution, pH 6 mg/mL eye/dose Days on Day 5(Control) 3 C 3 Compound-I; 4 30 μL/ QD for 4 1 hr post dose OphthalmicSolution, pH 6 mg/mL eye/dose Days on Day 5 (Control) 4 D 3 Formula II;4 30 μL/ QD for 4 1 hr post dose 0.08% HPMC + 2.5% mg/mL eye/dose Dayson Day 5 Glycerol + 0.2% HEC; 1.0 uM Vit K3; 30 μm particles, pH ~7.5 5E 3 Formula II; 6 30 μL/ QD for 4 1 hr post dose 0.08% HPMC + 2.5% mg/mLeye/dose Days on Day 5 Glycerol + 0.2% HEC; 1.0 uM Vit K3; 30 μmparticles, pH 6 6 F 3 Compound-I; 6 30 μL/ QD for 4 1 hr post dose 0.6%TRIS + 0.08% mg/mL eye/dose Days on Day 5 HPMC + 2.0% Glycerol + 0.2%HEC; 1.0 uM Vit K3; 30 μm particles, pH ~7.5 7 G 3 Compound-I; 4.3 30μL/ QD for 4 1 hr post dose 0.6% Tris + 2.0% mg/mL eye/dose Days on Day5 Glycerol + 0.3% HEC + 0.04% Tylox; 1.0 uM Vit K3; 30 μm particles, pH6 8 H 3 Compound-I; 4.3 30 μL/ QD for 4 1 hr post dose 0.6% Tris + 2.0%mg/mL eye/dose Days on Day 5 Glycerol + 0.2% HEC + 0.04% Tylox; 1.0 uMVit K3; 30 μm particles, pH 6 9 I 3 Compound-I; 4.3 30 μL/ QD for 4 1 hrpost dose 0.6% Tris + 2.0% mg/mL eye/dose Days on Day 5 Glycerol + 0.3%HEC + 0.04% Tylox; 1.0 uM Vit K3; 30 μm particles, pH 7 10 J 3Compound-I; 4.3 30 μL/ QD for 4 1 hr post dose 0.6% Tris + 2.0% mg/mLeye/dose Days on Day 5 Glycerol + 0.2% HEC + 0.04% Tylox; 1.0 uM Vit K3;30 μm particles, pH 7 11 K 3 Compound-I; 4.3 30 μL/ QD for 4 1 hr postdose 0.6% Tris + 2.0% mg/mL eye/dose Days on Day 5 Glycerol + 0.3% HEC +0.08% HPMC; 1.0 uM Vit K3; 30 μm particles, pH 6 12 L 3 Compound-I; 4.330 μL/ QD for 4 1 hr post dose 0.6% Tris + 2.0% mg/mL eye/dose Days onDay 5 Glycerol + 0.2% HEC + 0.08% HPMC; 1.0 uM Vit K3; 30 μm particles,pH 6 13 M 3 Compound-I; 4.3 30 μL/ QD for 4 1 hr post dose 0.6% Tris +2.0% mg/mL eye/dose Days on Day 5 Glycerol + 0.2% HEC + 0.08% HPMC; 1.0uM Vit K3; Native (~50 μm), pH 6 (Control) 14 N 3 Compound-I; 4.3 30 μL/QD for 4 1 hr post dose 0.6% Tris + 2.0% mg/mL eye/dose Days on Day 5Glycerol + 0.2% HEC + 0.08% HPMC; 1.0 uM Vit K3; 3 μm particles, pH 6 15O 3 Compound-I; 4.3 30 μL/ QD for 4 1 hr post dose 0.6% Tris + 2.0%mg/mL eye/dose Days on Day 5 Glycerol + 0.3% HEC + 0.08% HPMC; 1.0 uMVit K3; 30 μm particles, pH 7 16 P 3 Compound-I; 4.3 30 μL/ QD for 4 1hr post dose 0.6% Tris + 2.0% mg/mL eye/dose Days on Day 5 Glycerol +0.2% HEC + 0.08% HPMC; 1.0 uM Vit K3; 30 μm particles, pH 7 ^(a)A singledose will be administer on Day 5; QD - Once daily, BID - Twice daily

TABLE 18-2 Group # 1 2 3 4 5 6 7 8 9 Dose 0.3 1 4 4 6 6  4*  4*  4*strength (mg/mL) API Form Salt Salt Salt FB FB Salt Salt Salt SaltApprox. — — — 30/ 30/ 30/ 30/ 30/ 30/ PSD (μm) XL XL XL XL XL XL % — — —HPMC HPMC TRIS TRIS TRIS TRIS Stabilizer 0.08% 0.08% 0.60% 0.60% 0.60%0.60% % Glycerol — — — 2.5 2.5 2 2 2 2 % HEC — — — 0.2 0.2   0.2   0.3  0.2   0.3 Add'l — — — — — HPMC Tylox Tylox Tylox Excipient 0.08% 0.04%0.04% 0.04% VitK3 (μM) — — — 1 1 1 1 1 1 pH 6   6 6 ≈7.5 ≈7.5 6 6 6 7Frequency BID QD QD QD QD QD QD QD QD Control Group # 10 11 12 13 14 1516 Dose  4*  4*  4*  4*  4*  4*  4* strength (mg/mL) API Form Salt SaltSalt Salt Salt Salt Salt Approx. 30/ 30/ 30/ Native 3/ 30/ 30/ PSD (μm)XL XL XL (≈50)/ Large XL XL XXL % TRIS TRIS TRIS TRIS TRIS TRIS TRISStabilizer 0.60% 0.60% 0.60% 0.60% 0.60% 0.60% 0.60% % Glycerol 2 2 2 22 2 2 % HEC   0.2   0.3   0.2   0.2   0.2   0.3   0.2 Add'l Tylox HPMCHPMC HPMC HPMC HPMC HPMC Excipient 0.04% 0.08% 0.08% 0.08% 0.08% 0.08%0.08% VitK3 (μM) 1 1 1 1 1 1 1 pH 7 6 6 6 6 7 7 Frequency QD QD QD QD QDQD QD Control Suspension

Ocular sampling occurred one hour following the first daily dose on Day5 for all groups. Whole blood samples using K₂EDTA as an anticoagulantwere collected from all animals on Day 5, 1 hour post dose. The wholeblood was placed on ice until samples were spun in a centrifuge toseparate the plasma. Aqueous humor, conjunctiva, cornea, central retina,peripheral retina, central choroid, and peripheral choroid samples werecollected following necropsy. Samples were then frozen at −70° C. orlower. For all Groups 1-16, plasma, central retina, central choroid, andcornea samples were assayed for PAN-90806.

Ocular tissue concentrations of Formula II or Compound-I assessed on Day5 were in descending order: highest in the cornea>>centralchoroid>central retina. Corneal concentrations of Formula II orCompound-I ranged from 3 to 34 μM with the highest concentrations beingobserved in Group 9 and the lowest in Group 1. Central choroidconcentrations ranged from <0.056 to 0.24 μM with the highestconcentrations seen in Groups 6 and 14 and the lowest in Group 1.Central retina concentrations ranged from 0.02 to 0.14 μM; Group 6 hadthe highest central retina concentrations and Group 1 had the lowestcentral retina concentrations. Plasma concentrations of Formula II orCompound-I were very low being approximately 5 to 10-fold and 100-foldlower than those in the central retina and central choroid,respectively. See Table 18-3.

TABLE 18-3 Plasma Central Retina Central Choroid Cornea Group (μM) (μM)(μM) (μM) 1 <LLOQ 0.0163 <LLOQ 3.33 2 0.00267 0.0347 0.106 7.06 30.00450 0.0837 0.181 15.9 4 0.00327 0.0384 0.203 21.3 5 0.00391 0.04790.203 23.8 6 0.00998 0.136 0.243 14.4 7 0.00556 0.0697 0.142 9.68 80.00662 0.0901 0.152 11.3 9 0.00738 0.102 0.224 34.4 10 0.00528 0.05020.166 11.1 11 0.00465 0.0796 0.154 12.8 12 0.00366 0.0490 0.137 7.05 130.00524 0.0677 0.234 7.82 14 0.00499 0.0992 0.241 20.7 15 0.00325 0.05320.200 18.2 16 0.00523 0.0744 0.208 14.7 Plasma LLOQ = 0.00188 μM CentralRetina LLOQ = 0.00751 μM Central Choroid LLOQ = 0.0563 μM Cornea LLOQ =0.0188 μM

Gross ocular examinations using the Draize scale for scoring ocularirritation were performed on all animals prior to the first dose on eachday during the study. All formulations were well tolerated. In addition,animals were observed for general health pre-study, during dosing, andat necropsy. All animals assigned to study were normal. Necropsyevaluations consisted of observations in addition to those already notedon Draize Score sheets. For all formulations, dosing observations wereassessed using the Draize ocular irritation scoring system and aresummarized below. No abnormal observations were noted.

Example 20-4

Ocular Pharmacokinetics of Formulations of the Application in DutchBelted Rabbits

A non-GLP study was conducted to assess the ocular pharmacokinetics of afirst active agent of the present application (e.g., Formula II orCompound-I), in suspension, when administered one or two times daily, asa topical instillation, to both eyes of Dutch Belted rabbits for fourdays. The study design (Table 19-1) consisted of forty-seven (47) DutchBelted rabbits, each receiving a 30 μL bilateral topical ocular dose.Various suspension formulations of the present application wereevaluated and compared against a cyclodextrin-based eye drops solution(Control Group 1, Table 19-1). A comparison of the formulation variablesfor each suspension listed in Table 19-1 is provided in Table 19-2.Formula II or Compound-I was used in the test suspensions as well as theEye Drops solution. However, different batches of Compound-I were testedin certain suspensions (Groups 13, 14, and 15, Table 19-1).

TABLE 19-1 Dosing No. of Dose Frequency Sample Group Rabbits TestArticle Formulation Conc. Volume and Duration^(a) Time Points 1 A 3Compound-I; 4 30 μL/ QD for 4 1 hr post dose Ophthalmic Solution, mg/mLeye/dose days on Day 5 pH 6 (Control) 2 B 4 Compound-I; 4 30 μL/ QD for4 1 hr post dose HPMC; mg/mL eye/dose days on Day 5 30 μm particles 3 C3 Compound-I; 4 30 μL/ QD for 4 1 hr post dose HPMC; mg/mL eye/dose dayson Day 5 3 μm particles 4 D 4 Compound-I; 4 30 μL/ QD for 4 1 hr postdose HPMC; mg/mL eye/dose days on Day 5 50 μm particles (Control)  5* E3 Compound-I; 4 30 μL/ QD for 4 1 hr post dose HPMC; mg/mL eye/dose dayson Day 5 30 μm particles (Control)* 6 F 3 Compound-I; 4 30 μL/ BID for 41 hr post dose HPMC; mg/mL eye/dose days on Day 5 50 μm particles 7 G 3Compound-I; 1 30 μL/ QD for 4 1 hr post dose HPMC; mg/mL eye/dose dayson Day 5 50 μm particles 8 H 3 Compound-I; 1 30 μL/ BID for 4 1 hr postdose HPMC; mg/mL eye/dose days on Day 5 50 μm particles 9 I 3Compound-I; 2 30 μL/ QD for 4 1 hr post dose HPMC; mg/mL eye/dose dayson Day 5 50 μm particles 10  J 3 Compound-I; 2 30 μL/ BID for 4 1 hrpost dose HPMC; mg/mL eye/dose days on Day 5 50 μm particles 11  K 3Compound-I; 6 30 μL/ QD for 4 1 hr post dose HPMC; mg/mL eye/dose dayson Day 5 50 μm particles 12  L 3 Compound-I; 10 30 μL/ QD for 4 1 hrpost dose HPMC; mg/mL eye/dose days on Day 5 50 μm particles 13  M 3Compound-I; 4 30 μL/ QD for 4 1 hr post dose HPMC; mg/mL eye/dose dayson Day 5 50 μm particles 14  N 3 Compound-I; 4 30 μL/ QD for 4 1 hr postdose HPMC; mg/mL eye/dose days on Day 5 50 μm particles 15  O 3Compound-I; 4 30 μL/ QD for 4 1 hr post dose HPMC; mg/mL eye/dose dayson Day 5 50 μm particles ^(a)A single dose will be administer on Day 5*Sampled analyzed for Vitamin K3 QD - Once daily, BID - Twice daily

TABLE 19-2 Group # 1 2 3 4 5 6 7 N 3 4 2 4 3 3 3 Dose 4  4*  4*  4*  4* 4* 1 strength (mg/mL) Approx. — 30  3 Native Native Native Native PSD(≈50) (≈50) (≈50) (≈50) (μm) % — TRIS TRIS TRIS TRIS TRIS TRISStabilizer 0.60% 0.60% 0.60% 0.60% 0.60% 0.60% % — 2 2 2 2 2 2 Glycerol% HEC —   0.2   0.2   0.2   0.2   0.2   0.2 Add'l — HPMC HPMC HPMC HPMCHPMC HPMC Excip. 0.08% 0.08% 0.08% 0.08% 0.08% 0.08% Vitamin — 1 1 1 1 11 K3 (μM) pH 6 6 6 6 6 6 6 Dose QD QD QD QD QD BID QD Freq. ControlSuspension Suspension control control Group # 8 9 10 11 12 13 14 15 N 33 3 3 3 3 3 3 Dose 1 2 2 6 10   4*  4*  4* strength (mg/mL) Approx.Native Native Native Native Native Native Native Native PSD (≈50) (≈50)(≈50) (≈50) (≈50) (≈50) (≈50) (≈50) (μm) % TRIS TRIS TRIS TRIS TRIS TRISTRIS TRIS Stabilizer 0.60% 0.60% 0.60% 0.60% 0.60% 0.60% 0.60% 0.60% % 22 2 2 2 2 2 2 Glycerol % HEC   0.2   0.2   0.2   0.2   0.2   0.2   0.2  0.2 Add'l HPMC HPMC HPMC HPMC HPMC HPMC HPMC HPMC Excip. 0.08% 0.08%0.08% 0.08% 0.08% 0.08% 0.08% 0.08% Vitamin 1 1 1 1 1 1 1 1 K3 (μM) pH 66 6 6 6 6 6 6 Dose BID QD BID QD QD QD QD QD Freq. *4 mg/mL = 0.43% insalt form

Ocular sampling occurred one hour following the first daily dose on Day5 for all groups. Whole blood samples using K₂EDTA as an anticoagulantwere collected from all animals on Day 5, 1 hour post dose. The wholeblood was placed on ice until samples were spun in a centrifuge toseparate the plasma. Aqueous humor, conjunctiva, cornea, central retina,peripheral retina, central choroid, and peripheral choroid samples werecollected following necropsy. Samples were then frozen at −70° C. orlower. For all Groups 1-15, plasma, central retina, central choroid, andcornea samples were assayed. For Group 5 only, samples were analyzed forVitamin K3.

Ocular tissue concentrations of Formula II or Compound-I assessed on Day5 1 hour after the first dose were in descending order: highest in thecornea>>central choroid>central retina. Corneal concentrations ofFormula II or Compound-I ranged from 1 to 25 μM with the highestconcentrations being observed in Group 12 and the lowest in Group 7.Central choroid concentrations ranged from 0.06 to 0.2 μM with thehighest concentrations seen in Groups 12 and 14 and the lowest in Group7. Central retina concentrations ranged from 0.03 to 0.1 μM; Group 12had the highest central retina concentrations and Group 7 had the lowestcentral retina concentrations. Plasma concentrations of Formula II orCompound-I were very low being approximately 10-fold and 100-fold lowerthan those in the central retina and central choroid, respectively. SeeTable 19-3.

TABLE 19-3 Plasma Central Retina Central Choroid Cornea Group (μM) (μM)(μM) (μM) 1 0.00428 0.0551 0.129 14.3 2 0.00464 0.0506 0.135 7.48 30.00704 0.0928 0.159 10.2 4 0.00598 0.0735 0.128 3.93 5 0.00626 0.04420.129 4.47 6 0.00415 0.0441 0.199 6.41 7 0.00218 0.0256 0.0590 1.24 80.00211 0.0342 0.0708 1.72 9 0.00405 0.0634 0.0908 2.28 10 0.003760.0507 0.116 5.00 11 0.00772 0.0915 0.148 7.07 12 0.00861 0.114 0.19924.7 13 0.00709 0.0766 0.151 8.55 14 0.00268 0.0384 0.203 3.88 150.00469 0.0630 0.196 4.23 Plasma LLOQ = 0.00188 μM Central Retina LLOQ =0.00751 μM Central Choroid LLOQ = 0.0563 μM Cornea LLOQ = 0.0376 μM

An analytical method for the analysis of Vitamin K3 was determined inplasma, central retina, central choroid, and cornea samples from Group 5animals. A calibration curve prepared in control vitreous humor was usedto determine the concentration of Vitamin K3. Internal standard (IS)responses were inconsistent for the central choroid and plasma samples.Thus, positive plasma results were likely due to the reduced ISresponse. The Vitamin K3 response in plasma was similar to backgroundlevels. Central choroid samples had a positive Vitamin K3 response, butthe reduced IS response made quantitative results suspect. Centralretina and cornea samples had a normal IS response and no quantitativeresults for Vitamin K3. See Table 19-4.

TABLE 19-4 Plasma Central Retina Central Choroid Cornea Group (μM) (μM)(μM) (μM) 5 0.0923 <LLOQ 12.4 <LLOQ Plasma LLOQ = 0.0145 μM CentralRetina LLOQ = 0.581 μM Central Choroid LLOQ = 2.18 μM Cornea LLOQ =0.145 μM

Gross ocular examinations using the Draize scale for scoring ocularirritation were performed on all animals prior to the first dose on eachday during the study. In general, formulations were well tolerated, withthe exception of two animals in Group 6 on Day 4 of dosing, whereconjunctival chemosis was noted, one animal in Group 8 on Day 3, whereconjunctival discharge was noted, one animal in Group 10 on Day 5 ofdosing, where conjunctival chemosis and discharge were noted, one animalin Group 13, and one animal in Group 14 on Day 5 of dosing, whereconjunctival chemosis was noted. In addition, animals were observed forgeneral health pre-study, during dosing, and at necropsy. All animalsassigned to study were normal. Necropsy evaluations consisted ofobservations in addition to those already noted on Draize Score sheets.For all formulations, dosing observations were assessed using the Draizeocular irritation scoring system. The following clinical observationswere noted throughout the study: one animal in Group 8 exhibiteddischarge in the left eye during the dose on Day 3. Animals in Group 12exhibited chemosis in both eyes at the time of necropsy.

Example 21

Preparation of Particles Comprising Formula II

The suspension formulations comprising particles of Formula II wereprepared by roller milling. Large particles were produced using largermilling media (e.g., 3 mm in diameter) for a short period of time at lowroller speed (FIG. 9B). Nanoparticles were produced using smaller media(e.g., 0.8 mm in diameter) for a longer period of time at a high rollerspeed (FIG. 9A). The two concentrations of Formula II (i.e., 0.4% and0.2%) were diluted from a 5% API stock with a glycerol solution toobtain the proper tonicity. Best clean precautions included autoclavingof product contact parts, filtration of excipient solutions andtransfers using prepackaged sterile supplies. In addition, all transferswere within a laminar flow hood in a Clean Room area.

Additional examples of particles comprising Formula II or Compound-I isshown in Tables 21 and 22.

TABLE 21 Form. II or Formulation Cmpd-I Conc. API Form MethodDescription PSD  4 mg/mL Freebase Milled 0.08% HPMC, 2.5% Glycerol, 0.2%HEC, 1.0 uM 30 um Vitamin K3, pH 7.5  6 mg/mL Freebase Milled 0.08%HPMC, 2.5% Glycerol, 0.2% HEC, 1.0 uM 30 um Vitamin K3, pH 7.5  6 mg/mLHCl Salt Milled 0.6% Tris, 0.08% HPMC, 2.0% Glycerol, 0.2% 30 um HEC,1.0 uM Vitamin K3, pH 6.0 4.3 mg/mL HCl Salt Milled 0.6% Tris, 2.0%Glycerol, 0.3% HEC, 0.04% 30 um Tyloxapol, 1.0 uM Vitamin K3, pH 6.0 4.3mg/mL HCl Salt Milled 0.6% Tris, 2.0% Glycerol, 0.2% HEC, 0.04% 30 umTyloxapol, 1.0 uM Vitamin K3, pH 6.0 4.3 mg/mL HCl Salt Milled 0.6%Tris, 2.0% Glycerol, 0.3% HEC, 0.04% 30 um Tyloxapol, 1.0 uM Vitamin K3,pH 7.0 4.3 mg/mL HCl Salt Milled 0.6% Tris, 2.0% Glycerol, 0.2% HEC,0.04% 30 um Tyloxapol, 1.0 uM Vitamin K3, pH 7.0 4.3 mg/mL HCl SaltMilled 0.6% Tris, 2.0% Glycerol, 0.3% HEC, 0.08% 30 um HPMC, 1.0 uMVitamin K3, pH 6.0 4.3 mg/mL HCl Salt Milled 0.6% Tris, 2.0% Glycerol,0.2% HEC, 0.08% 30 um HPMC, 1.0 uM Vitamin K3, pH 6.0 4.3 mg/mL HCl SaltUn-milled 0.6% Tris, 2.0% Glycerol, 0.2% HEC, 0.08% Native ~50 HPMC, 1.0uM Vitamin K3, pH 6.0 um 4.3 mg/mL HCl Salt Milled 0.6% Tris, 2.0%Glycerol, 0.2% HEC, 0.08% 30 um HPMC, 1.0 uM Vitamin K3, pH 6.0 4.3mg/mL HCl Salt Milled 0.6% Tris, 2.0% Glycerol, 0.3% HEC, 0.08% 30 umHPMC, 1.0 uM Vitamin K3, pH 7.0 4.3 mg/mL HCl Salt Milled 0.6% Tris,2.0% Glycerol, 0.2% HEC, 0.08% 30 um HPMC, 1.0 uM Vitamin K3, pH 7.0

TABLE 22 Roller Milling PSD (Particle Size Distribution) Particles underAPI Form Excipient speed media size Median (nm) Mean (nm) D90 (nm)Storage Freebase Tween 80 ~300 ~2000 ~5400 Freebase Pluronic F-127 ~4700~5600 ~9000 Freebase Pluronic F-127 ~230 ~1000 ~2800 HCl salt Tyloxapol~110 ~110 ~160 Freebase Tween 80 low 3 mm ~5500 ~8400 ~11000 FreebaseTween 80 low 3 mm ~3900 ~4100 ~6800 Freebase Tween 80 low 3 mm ~3700~4000 ~7000 Freebase Tween 80 low 3 mm ~3500 ~4000 ~7600 FreebasePluronic F-127 low 3 mm ~6200 ~8100 ~12000 Freebase Pluronic F-127 low 3mm ~4400 ~4700 ~8100 Freebase Pluronic F-127 ~2500 ~3100 ~5800    6days, 40° C. HCl salt Tyloxapol low 3 mm ~6700 ~8500 ~16000 FreebasePluronic F-127 high 0.8 mm ~120 ~130 ~180  3 days, RT HCl salt PluronicF-127 high 0.8 mm ~100 ~100 ~140 HCl salt Tyloxapol high 0.8 mm ~110~120 ~170    6 days, 40° C. Freebase PVP K-29/32 high 0.8 mm ~150 ~210~290   1 day, RT Freebase PVP K-29/32 high 0.8 mm ~130 ~170 ~230 15days, RT HCl salt HPMC low 3 mm ~170 ~450 ~1200 21 days, RT FreebaseTween 80 high 0.8 mm ~160 ~370 ~1000 28 days, RT Freebase Pluronic F-127~3400 ~3700 ~6600

Example 22

Preparation of Compound-I/Tris Formulation

A non-milled suspension of the Compound-I was prepared. The formulationconsisted of: 3% Compound-I; 0.6% Tris-HCl pH 6; 2% Glycerol. Compound-Iwas added to the solution directly and mixed without milling andcharacterized (FIG. 9C).

Example 23

Particles Comprising Formula II and Vitamin K3

Addition of Vitamin K₃ (menadione) to the suspension formulation mayhave a beneficial in-vivo effect. Menadione was added to the formulationas a milled suspension. Menadione was initially milled exactly as theFormula II was milled; a 5% suspension with Pluronic F-127 as thestabilizer. This suspension was then added to Formula II before milling(large and nanoparticle formulations). The milled 5% suspensions(containing menadione suspension) were then diluted to 0.4% of FormulaII (in glycerol, as previously described).

Menadione did not appear to affect any physical characteristics of themilled particles of Formula II (FIG. 9D). However, at the highestconcentration of menadione added (10 μM), it was detected particles ofvitamin K₃ that were increasing in size. Additionally, there was a smallpopulation of very large particles outside the range of the free base,which was presumably the vitamin K₃. It was concluded that the vitaminK₃ could not be homogeneously distributed as well as the other particlesin the suspension, as they were growing at a faster rate than theFormula II.

Example 24

Preparation of Particles Comprising Menadione

Formulation of menadione/Formula II may form larger crystals over time.An optimized formulation was required to be able to add the menadione asa suspension. Formulations containing 5% menadione were screened using aroller mill in the same way as when screening the Formula IIformulations. An HPMC formulation produced a fine, homogeneoussuspension that demonstrated short term stability at 40° C. (FIG. 10).

Example 25

Preparation of Particles Comprising Formula II

The HPMC formulation for menadione was used with Formula II to provide aformulation in which the menadione could be added as a suspension. Itwas found that an X-large (30-40 micron) particle size using HPMC couldbe milled in a shorter time than when using Pluronic F-127 as thestabilizer (FIGS. 11A and 11B). The suspension could then be dilutedwith a menadione/HPMC suspension.

Example 25

Stability of Menadione in Formulations of the Present Application

The menadione concentration was 20 μM in the stability study. Themenadione was formulated as follows: 0.43% w/v Compound-I (unmilled, 50μm), 0.6% Tris/HCl, pH 6, 2% Glycerol, 0.2% HEC, 0.08% HPMC and 20 μMmenadione. From this formulation, ten test formulations were prepared:

1. minus glycerin (no N₂)

2. control formulation; (no N₂)

3. control formulation; N₂ sparged

4. +0.3% Na thiosulfate (pentahydrate), N₂ sparged*

5. +0.1% Na2 EDTA, N₂ sparged

6. +0.5% TPGS Vitamin E, N₂ sparged

7. +0.05% propyl gallate, N₂ sparged

8. +0.02% BHT, N₂ sparged

9. +menadione/HPb-cyclodextrin complex, N₂ sparged

10. +anti-oxidant complex (#5-#9), N₂ sparged

7 mL of each formulation was added to an amber 40 ml vial (under amberlighting). Filtered nitrogen gas was slowly bubbled into the bottom ofthe vial until the generated foam began to exit the top of the vial,which was then quickly capped. Menadione/HPb-Cyclodextrin complex (#9)was prepared by mixing approximately equimolar concentrated solutions ofcyclodextrin and menadione before addition (approximately 2 hours) tothe formulation.

Example 26

Methods for Roller Milling and Characterization of the Particles of thePresent Application Roller Mill

The horizontal roller mill (US Stoneware, model 755) consists of four,motor driven 12″ rubber rollers contained within a metal housing.Individual bottles placed between the rollers will rotate at an rpmdetermined by the speed of the rollers and the diameter of the bottle.Drug slurry consisting of API, stabilizers, water and milling media wasadded to the bottle and the cap tightly sealed before placing betweenthe rollers. The media used was an extremely dense Yttria Zirconium beadthat varies in diameter from 800 microns to 3000 microns. After milling,the dispersion was separated from the media by transferring the contentsto a centrifuge tube insert fitted with a screen mesh. The small insertwas placed into a centrifuge tube. The centrifuge was then run atapproximately 300×G for approximately 5 minutes. The dispersioncollected below the mesh (which retained the media) into the tube.

Optical Microscopy (OM)

Optical microscopy photomicrographs of nanoparticles were taken using anOlympus BX51 system equipped with an oil immersion 100× objective (1000×magnification). A calibration bar (from 1 um to 100 um) was set as acomparator on each photomicrograph. (The calibration bar effectivelyserves to size larger particles at lower magnifications, as well.)

Particle Size Distribution (PSD)

Particle size distribution was analyzed using laser diffraction lightscattering with a Horiba LA-950 V2. Generic assumptions were made insetting conditions and the refractive index value. The distributionswere volume based. Sample density was adjusted to a generic range ofpercent transmission on the blue LED light source. A small sample cell(filled with water) was used rather than the flow through cell, tominimize sample quantity.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes. In the present application the host document is identifiedwith sufficient particularity and materials that are relevant to thedisclosure are construed based on the context of the reference. Citationof publications and patent documents is not intended as an admissionthat any is pertinent prior art, nor does it constitute any admission asto the contents or date of the same. The application having now beendescribed by way of written description, those of skill in the art willrecognize that the application can be practiced in a variety ofembodiments and the foregoing description and examples are for purposesof illustration and not limitation of the claims that follow.

EQUIVALENTS

The application can be embodies in other specific forms withoutdeparting from the spirit or essential characteristics thereof. Theforegoing embodiments are therefore to be considered in all respectsillustrative rather than limiting on the application described herein.Scope of the application is thus indicated by the appended claims ratherthan by the foregoing description, and all changes that come within themeaning and range of equivalency of the claims are intended to beembraced therein.

The invention claimed is:
 1. A topical, ocular, suspension formulation,comprising: a. a first active agent of Formula II:

or a pharmaceutically acceptable salt thereof; b. a second active agent,or a pharmaceutically acceptable salt thereof, wherein the second activeagent is nicotinic acid, nicotinamide, or vitamin K, or a combinationthereof; and c. a pharmaceutically acceptable excipient; wherein thefirst active agent or the pharmaceutically acceptable salt thereof ispresent in about 0.1% to about 2.0% w/v.
 2. The formulation of claim 1,comprising Compound-I:


3. The formulation of claim 1, comprising about 0.1%, about 0.2%, about0.3%, or about 0.4% w/v of the compound of Formula II or apharmaceutically acceptable salt thereof.
 4. The formulation of claim 1,wherein the second active agent is vitamin K.
 5. The formulation ofclaim 1, comprising particles of the first active agent, or apharmaceutically acceptable salt thereof, wherein the particles have amean diameter of between 100 nm and 100 μm.
 6. The formulation of claim5, wherein the particles have a mean diameter of between 30 μm and 60μm.
 7. The formulation of claim 5, wherein the particles have a meandiameter of between 1 μm and 5 μm.
 8. The formulation of claim 5,wherein the particles have a mean diameter of at most 150 nm.
 9. Theformulation of claim 5, wherein the particles have a mean diameter ofabout 3 μm, about 30 μm, about 35 μm, or about 50 μm.
 10. Theformulation of claim 1, wherein the second active agent is present in anamount of less than 10 μM.
 11. The formulation of claim 10, wherein thesecond active agent is present in an amount of about 1 μM.
 12. Theformulation of claim 1, further comprising one or more excipientsselected from Polysorbate (Tween) 80, Poloxamer (Pluronic) F-127,Hypromellose (Hydroxypropyl Methylcellulose or HPMC), Povidone (PVPK-29/32 or K-30), and Tyloxapol, and a combination thereof.
 13. Theformulation of claim 1, comprising about 0.1% to about 1.0% (w/v) of thecompound of Formula II or a pharmaceutically acceptable salt thereof.14. The formulation of claim 1, comprising about 0.2% to about 1.0%(w/v) of the compound of Formula II or a pharmaceutically acceptablesalt thereof.
 15. The formulation of claim 1, comprising about 0.2%,about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%,about 0.9%, or about 1.0% (w/v) of the compound of Formula II or apharmaceutically acceptable salt thereof.
 16. The formulation of claim1, wherein the second active agent is present in an amount of about 1μM, about 2 μM, about 3 μM, about 4 μM, or about 5 μM.
 17. Theformulation of claim 1, further comprising a stabilizer for the secondactive agent.
 18. The formulation of claim 1, wherein the formulationhas a pH of about 6.